ISE Biology [13 ed.] 1265128847, 9781265128845

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Thirteenth Edition

Biology Kenneth A. Mason University of Iowa

Jonathan B. Losos

William H. Danforth Distinguished University Professor and Director, Living Earth Collaborative, Washington University

Tod Duncan

University of Colorado Denver

Based on the work of Peter H. Raven President Emeritus, Missouri Botanical Garden; George Engelmann Professor of Botany Emeritus, Washington University George B. Johnson Professor Emeritus of Biology, Washington University

BIOLOGY Published by McGraw Hill LLC, 1325 Avenue of the Americas, New York, NY 10019. Copyright ©2023 by McGraw Hill LLC. All rights reserved. Printed in the United States of America. No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of McGraw Hill LLC, including, but not limited to, in any network or other electronic storage or transmission, or broadcast for distance learning. Some ancillaries, including electronic and print components, may not be available to customers outside the United States. This book is printed on acid-free paper. 1 2 3 4 5 6 7 8 9 LWI 27 26 25 24 23 22 ISBN 978-1-265-12884-5 MHID 1-265-12884-7 Cover Image: Tree Frog: Shutterstock/Kurit afshen, Mushrooms: Russell Illig/Getty Images, Antibodies immunoglobulins attacking coronavirus: Corona Borealis Studio/Shutterstock

All credits appearing on page or at the end of the book are considered to be an extension of the copyright page. The Internet addresses listed in the text were accurate at the time of publication. The inclusion of a website does not indicate an endorsement by the authors or McGraw Hill LLC, and McGraw Hill LLC does not guarantee the accuracy of the information presented at these sites.

mheducation.com/highered

Brief Contents

Committed to Excellence xi Preparing Students for the Future xv

Part

I The Molecular Basis of Life 1

1 The Science of Biology  1 2 The Nature of Molecules and the Properties of Water  18 3 The Chemical Building Blocks of Life  35

Part 4 5 6 7 8 9 10

Part

II

Biology of the Cell 62

Cell Structure  62 Membranes 92 Energy and Metabolism  112 How Cells Harvest Energy  127 Photosynthesis 153 Cell Communication  175 How Cells Divide  194

III Genetic and Molecular Biology 216

11 Sexual Reproduction and Meiosis  216 12 Patterns of Inheritance  230 13 The Chromosomal Basis of Inheritance, and Human Genetics 248 14 DNA: The Genetic Material  268 15 Genes and How They Work  290 16 Control of Gene Expression  318 17 Biotechnology 341 18 Genomics 368 19 Cellular Mechanisms of Development  390

Part 20 21 22 23 24

Part

IV Evolution 416

Genes Within Populations  416 The Evidence for Evolution  443 The Origin of Species  463 Systematics, Phylogenies, and Comparative Biology  484 Genome Evolution  505

V Diversity of Life on Earth 524

25 The Origin and Diversity of Life  524 26 Viruses 539

27 28 29 30 31 32 33 34

Part 35 36 37 38 39 40

Part

Prokaryotes 560 Protists 586 Seedless Plants  610 Seed Plants  625 Fungi 643 Animal Diversity and the Evolution of Body Plans  666 Protostomes 688 Deuterostomes 721

VI Plant Form and Function 762

Plant Form  762 Transport in Plants  788 Plant Nutrition and Soils  807 Plant Defense Responses  825 Plant Sensory Systems  838 Plant Reproduction  866

VII Animal Form and Function 899

41 42 43 44 45 46 47 48 49 50 51 52

The Animal Body and Principles of Regulation  899 The Nervous System  923 Sensory Systems  953 The Endocrine System  979 The Musculoskeletal System  1003 The Digestive System  1023 The Respiratory System  1044 The Circulatory System  1063 Osmotic Regulation and the Urinary System  1085 The Immune System  1103 The Reproductive System  1133 Animal Development  1155

Part

VIII Ecology and Behavior 1185

53 54 55 56 57 58

Behavioral Biology  1185 Ecology of Individuals and Populations  1215 Community Ecology  1241 Dynamics of Ecosystems  1264 The Biosphere and Human Impacts  1288 Conservation Biology  1317

Appendix A Glossary G-1 Index I-1

iii

About the Authors

Kenneth Mason maintains an association with the University of Iowa, Department of Biology after having served

as a faculty member for eight years. His academic positions, as a teacher and researcher, include the faculty of the University of Kansas, where he designed and established the genetics lab, and taught and published on the genetics of pigmentation in amphibians. At Purdue University, he successfully developed and grew large introductory biology courses and collaborated with other faculty in an innovative biology, chemistry, and physics course supported by the National Science Foundation. At the University of Iowa, where his wife served as ©Kenneth Mason

president of the university, he taught introductory biology and human genetics. His honor society memberships include Phi Sigma, Alpha Lambda Delta, and, by vote of Purdue pharmacy students, Phi Eta Sigma Freshman Honors Society.

Jonathan Losos is the William H. Danforth Distinguished University Professor in the Department of Biology at Washington University and Director of the Living Earth Collaborative, a partnership between the university, the Saint Louis Zoo, and the Missouri Botanical Garden. Losos’s research has focused on studying patterns of adaptive radiation and evolutionary diversification in lizards. He is a member of the National Academy of Sciences, a fellow of the American Academy of Arts and Science, and the recipient of several awards, including the Theodosius Dobzhanksy and David Starr Jordan Prizes, the Edward Osborne Wilson Naturalist Award, and ©Jonathan Losos

the Daniel Giraud Elliot Medal, as well as receiving fellowships from the John Guggenheim and David and Lucile Packard Foundations. Losos has published more than 250 scientific articles and has written two books, Lizards in an Evolutionary Tree: Ecology and Adaptive Radiation of Anoles (University of California Press, 2009) and Improbable Destinies: Fate, Chance, and the Future of Evolution (Penguin-Random House, 2017). He is currently in the process of writing his next book, on scientific research on the ecology and evolution of domestic cats.

Tod Duncan is a Clinical Associate Professor at the University of Colorado Denver. He currently teaches the first semester of introductory biology, which focuses on molecular and cellular systems; he also teaches upperdivision courses in virology and cancer biology. Tod also runs the first-semester introductory biology labs in the course-based undergraduate research (CURE) format, in which up to 800 students each semester perform metagenomic analysis of purebred Labrador retriever gastrointestinal microbiota. Previously, he taught general microbiology, virology, the biology of cancer, medical microbiology, and cell biology. A bachelor’s degree in cell ©Lesley Howard

biology with an emphasis on plant molecular and cellular biology from the University of East Anglia in England led to doctoral studies in cell cycle control, and postdoctoral research on the molecular and biochemical mechanisms of DNA alkylation damage in vitro and in Drosophila melanogaster. Currently, he is interested in factors affecting retention and success of incoming first-year students in diverse demographics.

iv

Contents Committed to Excellence xi Preparing Students for the Future xv

Soames Summerhays/Natural Visions



Part

I The Molecular Basis

of Life

1 The Science of Biology 1

1.1 The Science of Life  2 1.2 The Nature of Science  4 1.3 An Example of Scientific Inquiry: Darwin and Evolution 8 1.4 Core Concepts in Biology  12



2 The Nature of Molecules and the Properties of Water 18

2.1 2.2 2.3 2.4 2.5 2.6

The Nature of Atoms  19 Elements Found in Living Systems  23 The Nature of Chemical Bonds  24 Water: A Vital Compound  26 Properties of Water  29 Acids and Bases  30

3 The Chemical Building Blocks of Life 35

3.1 Carbon: The Framework of Biological Molecules  36 3.2 Carbohydrates: Energy Storage and Structural Molecules 40 3.3 Nucleic Acids: Information Molecules  43 3.4 Proteins: Molecules with Diverse Structures and Functions 46 3.5 Lipids: Hydrophobic Molecules  56



Dr. Gopal Murti/Science Source



Part

II Biology of the Cell

4 Cell Structure 62

4.1 Cell Theory  63 4.2 Prokaryotic Cells  66 4.3 Eukaryotic Cells  68



4.4 The Endomembrane System  72 4.5 Mitochondria and Chloroplasts: Cellular Generators 76 4.6 The Cytoskeleton  78 4.7 Extracellular Structures and Cell Movement  82 4.8 Cell-to-Cell Interactions  85

5 Membranes 92

5.1 5.2 5.3 5.4 5.5 5.6

The Structure of Membranes  93 Phospholipids: The Membrane’s Foundation  96 Proteins: Multifunctional Components  98 Passive Transport Across Membranes  100 Active Transport Across Membranes  104 Bulk Transport by Endocytosis and Exocytosis  106

6 Energy and Metabolism 112

6.1 The Flow of Energy in Living Systems  113 6.2 The Laws of Thermodynamics and Free Energy  114 6.3 ATP: The Energy Currency of Cells  117 6.4 Enzymes: Biological Catalysts  118 6.5 Metabolism: The Chemical Description of Cell Function 122

7 How Cells Harvest Energy 127

7.1 Overview of Cellular Respiration  128 7.2 Glycolysis: Splitting Glucose  132 7.3 The Oxidation of Pyruvate Produces Acetyl-CoA 135 7.4 The Citric Acid Cycle  136 7.5 The Electron Transport Chain and Chemiosmosis 139 7.6 Energy Yield of Aerobic Respiration  142 7.7 Regulation of Aerobic Respiration  143 7.8 Oxidation Without O2 144 7.9 Catabolism of Proteins and Fats  146 7.10 Evolution of Metabolism  148

8 Photosynthesis 153

8.1 Overview of Photosynthesis  154 8.2 The Discovery of Photosynthetic Processes 155 8.3 Pigments 157 8.4 Photosystem Organization  160 8.5 The Light-Dependent Reactions  162 8.6 Carbon Fixation: The Calvin Cycle  166 8.7 Photorespiration 169

v

9 Cell Communication 175

9.1 9.2 9.3 9.4

Overview of Cell Communication  176 Receptor Types  179 Intracellular Receptors  180 Signal Transduction Through Receptor Kinases 182 9.5 Signal Transduction Through G Protein–Coupled Receptors 186



10 How Cells Divide 194

10.1 10.2 10.3 10.4 10.5

Bacterial Cell Division  195 Eukaryotic Chromosomes  197 Overview of the Eukaryotic Cell Cycle  200 Interphase: Preparation for Mitosis  201 M Phase: Chromosome Segregation and the Division of Cytoplasmic Contents  202 10.6 Control of the Cell Cycle  206 10.7 Genetics of Cancer  211



Steven P. Lynch

III Genetic and Molecular

Part

Biology

11 Sexual Reproduction and Meiosis 216

11.1 11.2 11.3 11.4

Sexual Reproduction Requires Meiosis  217 Features of Meiosis  218 The Process of Meiosis  219 Summing Up: Meiosis versus Mitosis  225

12 Patterns of Inheritance 230

12.1 The Mystery of Heredity  231 12.2 Monohybrid Crosses: The Principle of Segregation 233 12.3 Dihybrid Crosses: The Principle of Independent Assortment 236 12.4 Probability: Predicting the Results of Crosses  238 12.5 Extensions to Mendel  239

13 The Chromosomal Basis of Inheritance, and Human Genetics 248

13.1 Sex Linkage and the Chromosomal Theory of Inheritance 249 13.2 Exceptions to Mendelian Inheritance  251 13.3 Genetic Mapping  253 13.4 Human Genetics  256 13.5 Human Genetic Mapping and Association Studies  262

14 DNA: The Genetic Material 268

14.1 The Nature of the Genetic Material  269 14.2 DNA Structure  271

vi Contents



14.3 14.4 14.5 14.6

Basic Characteristics of DNA Replication  275 Prokaryotic Replication  278 Eukaryotic Replication  283 DNA Repair  285

15 Genes and How They Work 290

15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9

The Nature of Genes  291 The Genetic Code  294 Prokaryotic Transcription  297 Eukaryotic Transcription  299 Eukaryotic pre-mRNA Splicing  301 The Structure of tRNA and Ribosomes  303 The Process of Translation  306 Summarizing Gene Expression  310 Mutation: Altered Genes  311

16 Control of Gene Expression 318

16.1 16.2 16.3 16.4 16.5 16.6 16.7

Control of Gene Expression  319 Regulatory Proteins  320 Prokaryotic Regulation  322 Eukaryotic Regulation  326 Chromatin Structure Affects Gene Expression  329 Eukaryotic Posttranscriptional Regulation  331 Protein Degradation  335

17 Biotechnology 341

17.1 Recombinant DNA  342 17.2 Amplifying DNA Using the Polymerase Chain Reaction 346 17.3 Creating, Correcting, and Analyzing Genetic Variation 349 17.4 Constructing and Using Transgenic Organisms  351 17.5 Environmental Applications  355 17.6 Medical Applications  357 17.7 Agricultural Applications  361

18 Genomics 368

18.1 18.2 18.3 18.4 18.5 18.6

Mapping Genomes  369 Sequencing Genomes  372 Genome Projects  375 Genome Annotation and Databases  377 Comparative and Functional Genomics  380 Applications of Genomics  385

19 Cellular Mechanisms of Development 390

19.1 The Process of Development  391 19.2 Cell Division  391 19.3 Cell Differentiation  393 19.4 Nuclear Reprogramming  398 19.5 Pattern Formation  402 19.6 Evolution of Pattern Formation  407 19.7 Morphogenesis 410

tamoncity/Shutterstock

IV

Part



Evolution

20 Genes Within Populations 416

20.1 20.2 20.3 20.4 20.5 20.6 20.7

Genetic Variation and Evolution  417 Changes in Allele Frequency  418 Five Agents of Evolutionary Change  420 Quantifying Natural Selection  425 Reproductive Strategies  426 Natural Selection’s Role in Maintaining Variation  430 Selection Acting on Traits Affected by Multiple Genes 432 20.8 Experimental Studies of Natural Selection  434 20.9 Interactions Among Evolutionary Forces  436 20.10 The Limits of Selection  437

21 The Evidence for Evolution 443

21.1 The Beaks of Darwin’s Finches: Evidence of Natural Selection 444 21.2 Peppered Moths and Industrial Melanism: More Evidence of Selection  446 21.3 Artificial Selection: Human-Initiated Change  448 21.4 Fossil Evidence of Evolution  450 21.5 Anatomical Evidence for Evolution  454 21.6 Convergent Evolution and the Biogeographical Record 456 21.7 Darwin’s Critics  458

22 The Origin of Species 463

22.1 The Nature of Species and the Biological Species Concept 464 22.2 Natural Selection and Reproductive Isolation  468 22.3 The Role of Genetic Drift and Natural Selection in Speciation 470 22.4 The Geography of Speciation  471 22.5 Adaptive Radiation and Biological Diversity  473 22.6 The Pace of Evolution  478 22.7 Speciation and Extinction Through Time  479

23 Systematics, Phylogenies, and Comparative Biology 484

23.1 Systematics 485 23.2 Cladistics 486 23.3 Systematics and Classification  490 23.4 Phylogenetics and Comparative Biology  493 23.5 Phylogenetics and Disease Evolution  499

Jeff Hunter/The Image Bank/Getty Images



Part

V Diversity of Life

on Earth

25 The Origin and Diversity of Life 524

25.1 25.2 25.3 25.4 25.5

Deep Time  526 Origins of Life  526 Evidence for Early Life  529 Earth’s Changing System  531 Ever-Changing Life on Earth  532

26 Viruses 539

26.1 26.2 26.3 26.4 26.5

The Nature of Viruses  540 Viral Diversity  544 Bacteriophage: Bacterial Viruses  546 Viral Diseases of Humans  547 Prions and Viroids: Infectious Subviral Particles  555

27 Prokaryotes 560

27.1 27.2 27.3 27.4 27.5 27.6

Prokaryotic Diversity  561 Prokaryotic Cell Structure  565 Prokaryotic Genetics  569 The Metabolic Diversity of Prokaryotes  574 Microbial Ecology  576 Bacterial Diseases of Humans  578

28 Protists 586

28.1 28.2 28.3 28.4 28.5 28.6 28.7 28.8 28.9

Eukaryotic Origins and Endosymbiosis  587 Overview of Protists  589 Characteristics of the Excavata  591 Characteristics of the SAR: Stramenopila  594 Characteristics of the SAR: Alveolata  596 Characteristics of the SAR: Rhizaria  600 Characteristics of the Archaeplastida  601 Characteristics of the Amoebozoa  604 Characteristics of the Opisthokonta  606

29 Seedless Plants 610

24 Genome Evolution 505







24.1 Comparative Genomics  506 24.2 Genome Size  509 24.3 Evolution Within Genomes  513

24.4 Gene Function and Expression Patterns  516 24.5 Applying Comparative Genomics  517



29.1 Origin of Land Plants  611 29.2 Bryophytes Have a Dominant Gametophyte Generation 613 29.3 Tracheophytes Have a Dominant Sporophyte Generation 615 29.4 Lycophytes Diverged from the Main Lineage of Vascular Plants  618 29.5 Pterophytes Are the Ferns and Their Relatives  619 Contents vii

30 Seed Plants 625

30.1 The Evolution of Seed Plants  626 30.2 Gymnosperms: Plants with “Naked Seeds”  626 30.3 Angiosperms: The Flowering Plants  630 30.4 Seeds 636 30.5 Fruits 637

31 Fungi 643

31.1 31.2 31.3 31.4 31.5 31.6 31.7 31.8 31.9

Classification of Fungi  644 Fungal Forms, Nutrition, and Reproduction  645 Fungal Ecology  648 Fungal Parasites and Pathogens  652 Basidiomycota: The Club (Basidium) Fungi  654 Ascomycota: The Sac (Ascus) Fungi  656 Glomeromycota: Asexual Plant Symbionts  658 Zygomycota: Zygote-Producing Fungi  658 Chytridiomycota and Relatives: Fungi with Zoospores 660 31.10 Microsporidia: Unicellular Parasites  661

32 Animal Diversity and the Evolution of Body Plans 666

32.1 32.2 32.3 32.4

Some General Features of Animals  667 Evolution of the Animal Body Plan  668 Animal Phylogeny  672 Parazoa: Animals That Lack Specialized Tissues 676 32.5 Eumetazoa: Animals with True Tissues  679 32.6 The Bilateria  684

33 Protostomes 688

33.1 33.2 33.3 33.4 33.5 33.6 33.7 33.8 33.9

The Clades of Protostomes  689 Flatworms (Platyhelminthes)  690 Rotifers (Rotifera)  693 Mollusks (Mollusca)  694 Annelids (Annelida)  700 Ribbon Worms (Nemertea)  703 Bryozoans (Bryozoa) and Brachiopods (Brachiopoda)  704 Roundworms (Nematoda)  706 Arthropods (Arthropoda)  708

34 Deuterostomes 721

34.1 Echinoderms 722 34.2 Chordates 724 34.3 Nonvertebrate Chordates  726 34.4 Vertebrate Chordates  727 34.5 Fishes 729 34.6 Amphibians 734 34.7 Reptiles 738 34.8 Birds 743 34.9 Mammals 747 34.10 Evolution of the Primates  752

viii Contents

Susan Singer/McGraw Hill



Part

VI Plant Form and

Function

35 Plant Form 762

35.1 35.2 35.3 35.4 35.5

Organization of the Plant Body: An Overview  763 Plant Tissues  766 Roots: Anchoring and Absorption Structures  772 Stems: Support for Above-Ground Organs  776 Leaves: Photosynthetic Organs  781

36 Transport in Plants 788

36.1 36.2 36.3 36.4 36.5 36.6

Transport Mechanisms  789 Water and Mineral Absorption  792 Xylem Transport  795 Rate of Transpiration  797 Water-Stress Responses  799 Phloem Transport  801

37 Plant Nutrition and Soils 807

37.1 Soils: The Substrates on Which Plants Depend  808 37.2 Plant Nutrients  811 37.3 Special Nutritional Strategies  813 37.4 Carbon–Nitrogen Balance and Global Change  816 37.5 Phytoremediation 819

38 Plant Defense Responses 825

38.1 38.2 38.3 38.4

Physical Defenses  826 Chemical Defenses  828 Animals That Protect Plants  831 Systemic Responses to Invaders  832

39 Plant Sensory Systems 838

39.1 39.2 39.3 39.4 39.5

Responses to Light  839 Responses to Gravity  843 Responses to Mechanical Stimuli  845 Responses to Water and Temperature  847 Hormones and Sensory Systems  849

40 Plant Reproduction 866

40.1 Reproductive Development  867 40.2 Making Flowers  869 40.3 Structure and Evolution of Flowers  874 40.4 Pollination and Fertilization  877 40.5 Embryo Development  882 40.6 Germination 888 40.7 Asexual Reproduction  891 40.8 Plant Life Spans  893

©Dr. Roger C. Wagner, Professor Emeritus of Biological Sciences, University of Delaware



Part

VII Animal Form and

Function

41 The Animal Body and Principles of Regulation 899

41.1 Organization of Animal Bodies  900 41.2 Epithelial Tissue  901 41.3 Connective Tissue  904 41.4 Muscle Tissue  907 41.5 Nerve Tissue  908 41.6 Overview of Vertebrate Organ Systems  909 41.7 Homeostasis 912 41.8 Regulating Body Temperature  914

42 The Nervous System 923

42.1 Nervous System Organization  924 42.2 The Mechanism of Nerve Impulse Transmission 927 42.3 Synapses: Where Neurons Communicate with Other Cells 932 42.4 The Central Nervous System: Brain and Spinal Cord  938 42.5 The Peripheral Nervous System: Spinal and Cranial Nerves 945

43 Sensory Systems 953

43.1 43.2 43.3 43.4 43.5

Overview of Sensory Receptors  954 Touch, Pressure, and Body Position  955 Hearing, Vibration, and Balance  957 Taste, Smell, and pH  963 Temperature, Pain, Electric Currents, and Magnetic Fields 965 43.6 Vision 967 43.7 Evolution and Development of Eyes  973

44 The Endocrine System 979

44.1 Regulation of Body Processes by Chemical Messengers  980 44.2 Overview of Hormone Action  985 44.3 The Pituitary and Hypothalamus: The Body’s Control Centers 988 44.4 The Major Peripheral Endocrine Glands  993 44.5 Other Hormones and Their Effects  997

45 The Musculoskeletal System 1003

45.1 Types of Skeletal Systems  1004 45.2 A Closer Look at Bone  1006 45.3 Joints 1009 45.4 Muscle Contraction  1010 45.5 Vertebrate Skeleton Evolution and Modes of Locomotion  1017

46 The Digestive System 1023

46.1 Types of Digestive Systems  1024 46.2 The Mouth and Teeth: Food Capture and Bulk Processing 1026 46.3 The Esophagus and the Stomach: The Early Stages of Digestion  1027 46.4 The Intestines: Breakdown, Absorption, and Elimination 1029 46.5 Accessory Organ Function  1032 46.6 Neural and Hormonal Regulation of the Digestive Tract 1034 46.7 Food Energy, Energy Expenditure, and Essential Nutrients 1035 46.8 Variations in Vertebrate Digestive Systems  1039

47 The Respiratory System 1044

47.1 Gas Exchange Across Respiratory Surfaces  1045 47.2 Gills, Cutaneous Respiration, and Tracheal Systems 1046 47.3 Lungs 1049 47.4 Structures, Mechanisms, and Control of Ventilation in Mammals  1052 47.5 Transport of Gases in Body Fluids  1056

48 The Circulatory System 1063

48.1 48.2 48.3 48.4

Invertebrate Circulatory Systems  1064 The Components of Vertebrate Blood  1065 Vertebrate Circulatory Systems  1068 Cardiac Cycle, Electrical Conduction, ECG, and Cardiac Output  1071 48.5 Blood Pressure and Blood Vessels  1075

49 Osmotic Regulation and the Urinary System 1085

49.1 Osmolarity and Osmotic Balance  1086 49.2 Nitrogenous Wastes: Ammonia, Urea, and Uric Acid  1087 49.3 Osmoregulatory Organs  1088 49.4 Evolution of the Vertebrate Kidney  1090 49.5 The Mammalian Kidney  1092 49.6 Hormonal Control of Osmoregulatory Functions  1097

50 The Immune System 1103

50.1 50.2 50.3 50.4 50.5 50.6 50.7

Innate Immunity  1104 Adaptive Immunity  1109 Cell-Mediated Immunity  1114 Humoral Immunity and Antibody Production  1117 Autoimmunity and Hypersensitivity  1123 Antibodies in Medical Treatment and Diagnosis  1125 Pathogens That Evade the Immune System  1127

51 The Reproductive System 1133

51.1 Animal Reproductive Strategies  1134 51.2 Vertebrate Fertilization and Development  1136 Contents ix



51.3 Structure and Function of the Human Male Reproductive System  1140 51.4 Structure and Function of the Human Female Reproductive System  1144 51.5 Contraception and Infertility Treatments  1148



52 Animal Development 1155

52.1 Fertilization 1156 52.2 Cleavage and the Blastula Stage  1160 52.3 Gastrulation 1162 52.4 Organogenesis 1166 52.5 Vertebrate Axis and Pattern Formation  1171 52.6 Human Development  1178

K. Ammann/Bruce Coleman Inc./Photoshot



Part

VIII Ecology and

Behavior

53 Behavioral Biology 1185

53.1 The Natural History of Behavior  1186 53.2 Nerve Cells, Neurotransmitters, Hormones, and Behavior 1187 53.3 Behavioral Genetics  1188 53.4 Learning 1190 53.5 The Development of Behavior  1191 53.6 Animal Cognition  1194 53.7 Orientation and Migratory Behavior  1195 53.8 Animal Communication  1197 53.9 Behavior and Evolution  1200 53.10 Behavioral Ecology  1201 53.11 Reproductive Strategies  1204 53.12 Altruism  1206 53.13 The Evolution of Group Living and Animal Societies 1210

54 Ecology of Individuals and Populations 1215

54.1 The Environmental Challenges  1216 54.2 Populations: Groups of a Single Species in One Place 1218 54.3 Population Demography and Dynamics  1222

x Contents



54.4 54.5 54.6 54.7 54.8

Life History and the Cost of Reproduction  1224 Environmental Limits to Population Growth  1227 Factors That Regulate Populations  1229 Human Population Growth  1232 Pandemics and Human Health  1236

55 Community Ecology 1241

55.1 55.2 55.3 55.4 55.5

Biological Communities: Species Living Together  1242 The Ecological Niche Concept  1243 Predator–Prey Relationships  1248 The Many Types of Species Interactions  1252 Ecological Succession, Disturbance, and Species Richness 1258

56 Dynamics of Ecosystems 1264

56.1 56.2 56.3 56.4 56.5

Biogeochemical Cycles  1265 The Flow of Energy in Ecosystems  1271 Trophic-Level Interactions  1276 Biodiversity and Ecosystem Stability  1280 Island Biogeography  1283

57 The Biosphere and Human Impacts 1288

57.1 57.2 57.3 57.4 57.5

Ecosystem Effects of Sun, Wind, and Water  1289 Earth’s Biomes  1293 Freshwater Habitats  1296 Marine Habitats  1299 Human Impacts on the Biosphere: Pollution and Resource Depletion 1303 57.6 Human Impacts on the Biosphere: Climate Change 1309

58 Conservation Biology 1317

58.1 58.2 58.3 58.4

Overview of the Biodiversity Crisis  1318 The Value of Biodiversity  1322 Factors Responsible for Extinction  1325 An Evolutionary Perspective on the Biodiversity Crisis 1335 58.5 Approaches for Preserving Endangered Species and Ecosystems 1338

Appendix A Glossary G-1 Index I-1

Committed to Excellence With the new 13th edition, Raven and Johnson’s Biology continues the momentum built over the last five editions. We continue to pro­ vide an unmatched comprehensive text fully integrated with a continuously evolving, state-of-the-art digital environment. We remain committed to our roots as the majors biology text that best integrates evolution throughout the text. There is an emphasis on the relevance of evolution throughout the ecology section, not only in all four ecology chapters, but also in the chapters on behavioral and conservation biology. In the animal form and function section, we emphasize evolution in the context of physiology. We have also moved the examples and insights from the chapter devoted to the evolution of development, to place them into the appropriate contexts throughout the book. This emphasizes the importance of evolution and develop­ment by continually providing examples, rather than gathering them together in a single chapter. In the opening molecular chapters, we have added additional examples of the action of evolution at the molecular level. We have also renewed our commitment to the ideas set forth in the Vision and Change report from the AAAS, which provides a framework for modern undergraduate biology education. This re­port is now more than a decade old, yet still retains relevance. Perhaps the most important idea articulated by Vision and Change was an ­emphasis on core concepts. This emphasis is important because it is integral to how experts organize information in their brains. This allows an expert to incorporate new information more readily than a novice, who lacks an adequate conceptual framework. We have designed a new Visual Outline for each chapter opening and a more detailed Visual Summary at the end of each chapter. These provide a visual representation of one way an expert organizes the main concepts of a chapter, emphasizing connections and hierarchies of concepts. These are intended not to be exhaustive roadmaps of every chapter, but to show how important concepts for each chapter can be organized and connected. The presentation will appeal to students who are visual learners, and allow them to “see” the chapter as a whole. We encourage students to create their own visual maps of how the important concepts in each chapter are interconnected. One unanticipated consequence of the Vision and Change movement was that publishers chasing new approaches began to produce books so “feature-laden” as to be virtually unreadable by the average student. This continues to be an issue with many textbooks on the market. We have not abandoned the idea that narra­ tive flow is important, even in a science textbook. While we include a variety of features to improve student learning, they are integrated into the text and are not included at the expense of the concise, ac­cessible, and engaging writing style we are known for. A priority of each revision is to assess each chapter for clarity and

readability. With each revision, we strive to extend ­ the clear ­emphasis on evolution and scientific inquiry that have made this a leading textbook of choice for majors biology students. Faculty want a textbook that emphasizes both student-­ centered ap­proaches and core concepts for the biological sciences. As a team, we continually strive to improve the text by integrating the latest cognitive and best practices research with methods that are known to positively affect learning. We emphasize scientific ­inquiry, and have increased the quantitative component in the ­Scientific Thinking figures, as well as in the Inquiry and Data Analysis ­questions. Our text continues to be a leader with an organization that emphasizes important biological concepts, while keeping the s­ tudent engaged with learning outcomes that allow as­sessment of progress in understanding these concepts. An inquiry-based approach with robust, adaptive tools for discovery and assessment in both text and digital resources provides the intellec­tual challenge needed to promote student critical thinking and en­sure academic success. We continue to use our digital environment in the revision of Biology. A major strength of both text and digital resources is assess­ ment across multiple levels of Bloom’s taxonomy that develops critical-thinking and problem-solving skills in addition to com­ prehensive factual knowledge. McGraw Hill Connect® offers a powerful suite of online tools that are linked to the text and in­cludes quantitative assessment tools. The Data and Graphing Interactive exercises have been expanded for the 13th edition. This valuable digital tool uses data, controlled by the students, to engage them in actively exploring quantitative aspects of biology. Our adaptive SmartBook 2.0 learning system helps students learn faster, study efficiently, and retain more knowledge of key concepts. The 13th edition continues to employ the aesthetically stunning art program that the Raven and Johnson Biology text is known for. Complex topics are represented clearly and suc­cinctly, helping students build the mental models needed to understand biology. We’re excited about the 13th edition of this quality textbook providing a learning path for a new generation of students. All of us have extensive experience teaching undergraduate biology, and we’ve used this knowledge as a guide in producing a text that is up to date, beautifully illustrated, and pedagogically sound for the ­stu­dent. We are also excited about the continually evolving digital environment that provides a unique and engaging learning environ­ ment for modern students. We’ve worked hard to provide clear, explicit learning outcomes that closely integrate the text with its media support materials to provide instructors with an excellent complement to their teaching.

Ken Mason, Jonathan Losos, Tod Duncan

 xi

Cutting Edge Science Changes to the 13th Edition We authors started work on this new edition at the beginning of an unprecedented pandemic that has affected the entire world. In addition to the personal and professional upheaval that all have experienced, we were faced with the challenge of how to respond to the pandemic in this new edition. Given that this pandemic is primarily a biological phenomenon, we felt that it was important to include accurate information, but not to let the book be overwhelmed by this single event. So, we have included new material on the disease COVID-19, the virus SARS-CoV-2, and the ways scientists have analyzed and responded to this new pathogen. Rather than have a single section on this, we have included material throughout the book wherever it is relevant, including information about the virus itself, diagnostic methods, vaccines, evolution, and the population dynamics and environmental origin of epidemic diseases.

Part I: The Molecular Basis of Life Chapter 1—Edited for clarity and readability for the student. The Scientific Thinking figure was completely redone. Chapter 2—Minor edits for clarity, especially regarding hydrogen bonding and water. Chapter 3—Minor edits for clarity, especially regarding the structure of nucleic acids and proteins. Several figures were corrected for inaccuracies or ambiguities. Section on the role of trans fats was rewritten for clarity and currency.

Part II: Biology of the Cell Chapter 4—The entire section on prokaryotic cell structure was rewritten to take into account new data. This provides a different, and more modern, view of prokaryotic cell structure. A new figure comparing bacterial and archaeal flagella was added. Chapter 5—Minor edits for clarity and readability for the student. Chapter 6—Minor edits for clarity and readability for the student. The speculative material on evolution of metabolism was removed. Chapter 7—Minor edits for clarity and readability for the student. Chapter 8—Some editing for clarity. Also, the section on the experimental history of photosynthesis was rewritten. This reduced the amount of material while still providing students an historical and experimental context for the rest of the chapter. Chapter 9—Edited for clarity and to update some material for currency. Chapter 10—The section on chromosome structure was rewritten to take into account new data on the organization of chromatin in the nucleus. This complements the updates to the last edition, and

xii Committed to Excellence

includes a new figure showing the organization of chromosomes in the nucleus.

Part III: Genetic and Molecular Biology We reorganized the two chapters on transmission genetics to provide a more logical flow of concepts, and to emphasize modern human genetics. Chapter 11—Edited for clarity and readability for the student. Chapter 12—This chapter was entirely rewritten to bring a more modern focus. The history was retained to provide context for the entire chapter. Mendel’s model is presented in a clearer, more modern form. The extensions to Mendel were reorganized and also given a more modern perspective. Two new figures were added to replace older figures. Chapter 13—This chapter was completely rewritten to form a more cohesive whole with chapter 12, on Mendelian genetics. It begins with an updated treatment of the chromosomal theory of inheritance, which complements and extends chapter 12. The remainder of the chapter is devoted to a much more modern view of human genetics. Material on human genetics from the old chapter 12 was moved to this chapter and substantial additions were included to keep pace with the rapid pace of change in human genetics. Chapter 14—The material on DNA structure was rewritten to make some difficult concepts more clear to students. Chapter 15—The material on alternative splicing was updated. The section on mutation was completely rewritten to take new data into account, and to include current information on genetic variation. Chapter 16—The chapter was edited for clarity and currency. Chapter 17—The chapter was revised to include new and relevant technologies highlighted by the recent pandemic of COVID-19. There is a description of PCR-based tests for pathogens, and updated material on antibody-based tests to detect pathogens and exposure to pathogens. There is also a section on new vaccine technologies, including mRNA and subunit vaccines. Chapter 18—The sections on genome projects and annotation were both rewritten to take new data into account. These provide as current a view of these very fast-moving areas of biology as is possible for an introductory text. Chapter 19—Minor edits for clarity and readability for some difficult concepts.

Part IV: Evolution Chapter 20—The section on genetic variation in populations was extensively revised reflecting new information based on widespread genomic investigation. Genomic variation in humans is

now discussed in great detail, quantifying the extent of variation that exists and how that variation is apportioned within and between populations. Chapter 22—New information on the genes involved in the evolution of the beaks of Darwin’s finches was included. Chapter 23—Discussion on the evolution of the HIV virus was revised to include new discoveries and trends. An entirely new section was added detailing how phylogenetic analysis has helped track the evolution and spread of the virus causing COVID-19. The drawing of Archaeopteryx was revised to correspond to new analyses of fossils indicating that these early birds were dark in color. Chapter 24—The comparative genomics section was rewritten to emphasize the evolution of the vertebrate genome. The material on primate genome evolution was updated to reflect new data.

Part VI: Plant Form and Function There have been no major changes in the plant form and function chapters. There has been overall editing for readability and in response to recommendations by reviewers and users of the 12th edition.

Part VII: Animal Form and Function Chapter 41—Minor edits for clarity in the material on homeostasis and thermoregulation. Chapter 42—Minor edits for clarity and accuracy. Chapter 43—This chapter was reorganized to reflect the relationship among different types of receptors. The depiction of the evolution of the inner ear was revised to illustrate the transition from reptilian to mammalian hearing structures. Treatment of the phylogenetic analysis of the evolution of genes related to eye structure was modified in light of new discoveries.

Part V: Diversity of Life on Earth

Chapter 44—Minor edits for clarity and readability in material on receptor function.

Chapter 25—The material on supergroups was updated, including a new figure.

Chapter 45—Illustration of convergent evolution in skeletal structure of birds, bats, and pterosaurs was revised.

Chapter 26—A new section was added to provide students with contemporary information about the coronaviruses. Particular emphasis was placed on SARS-CoV-2, the causative agent of the COVID-19 pandemic. A new figure was added to help explain the infectious cycle of coronaviruses in general. The chapter was edited for readability.

Chapter 47—Discussion of external gills and respiration in salamanders was revised to reflect the variation in structures found within this order.

Chapter 27—This chapter was edited to improve readability. Epidemiological data on infectious diseases was updated to reflect changes since the 12th edition. Chapter 28—This chapter was completely restructured and large parts were rewritten to reflect current thinking in the taxonomy of the protists. Additional edits were made for readability and comprehension. Chapter 31—Edited for clarity and readability. Chapter 32—Discussion of relationships at the base of the phylogeny for all animals was revised to reflect new understanding and debate about relationships among sponges, ctenophores, and other animals. Additional changes were made to reflect other changes in understanding of phylogenetic relationships among animal taxa, such as the position of chaetognaths. Aspects of taxonomy and natural history were updated in line with new findings. Chapter 33—This chapter was revised and reorganized to reflect new understanding of relationships among protostome taxa. Chapter 34—Discussion of human evolution was revised in light of new discoveries.

Chapter 50—Much of the chapter was rewritten to improve the clarity and flow of ideas and emphasize connections between innate and adaptive immunity. A new section on vaccination was added to complement the new section in chapter 17 on vaccine technology. Chapter 51—Discussion of variation in reproductive systems among animals was revised with regard to parthenogenesis and reproductive mode. Chapter 52—Edited for clarity and readability of difficult concepts.

Part VIII: Ecology and Behavior Chapter 53—Discussions of behavioral genetics, migration, and other topics were revised to incorporate new findings on the genetic basis of behavior. Chapter 54—A new section was added on pandemics and human health that covers the general topic, but extensively details the population biology of the COVID-19 pandemic. Human population trends and other timely data were updated to stay current. Chapter 57—The section on deforestation was greatly updated with new information and trends, and a section on the effect of massive wildfires was added. Discussion of zoonotic diseases was increased, with emphasis on COVID-19. New discussion of

Committed to Excellence xiii

microplastics in the environment was added. The section on El Niño events was revised. All of the data on biosphere impacts of humans were updated to stay current. Chapter 58—Figures on the rate at which species are becoming endangered were updated and a section on the new-found data on decline in insects was added. Data on the relationship between human population growth and biodiversity decline was updated, as was information on the rate of establishment of invasive species. Information on case studies throughout the chapter was also updated to reflect the current situation.

A Note From the Authors A revision of this scope relies on the talents and efforts of many people working behind the scenes and we have benefited greatly from their assistance. Beth Bulger was the copy editor for this edition. She has ­labored many hours and always improves the clarity and consistency of the text. She has made significant contributions to the quality of the final product. We were fortunate to work again with MPS to update the art program and improve the layout of the pages. Our close collaboration resulted in a text that is pedagogically effective as well as more beautiful than any other biology text on the market. We have the continued support of an excellent team at McGraw Hill Education. Ian Townsend, Portfolio Manager for Biology, has been a steady leader during a time of change. Senior Product Developer Liz Sievers, provided support in so many ways it would be impossible to name them all. Kelly Hart, Content Project Manager, and David Hash, Designer, ensured our text was on time and elegantly designed. Kelly Brown, Senior Marketing Manager, is always a sounding board for more than just marketing, and many more people behind the scenes have all contributed to the success of our text. This includes Sarah Hosch and Elizabeth Drumm who greatly helped develop the new Visual Outlines and Visual Summaries, and the digital team, to whom we owe a great deal for their efforts to continue improving our Connect assessment tools. The digital Subject Matter Experts for this edition include: Faye Nourollahi, Austin Dreyer, Cynthia H. Dadmun, Timothy Hadlock, Elizabeth Drumm, and Lauri Carey. Throughout this edition we have had the support of spouses and families, who have seen less of us than they might have liked because of the pressures of getting this revision completed. They have adapted to the many hours this book draws us away from them, and, even more than us, looked forward to its completion.

xiv Committed to Excellence

In the end, the people we owe the most are the generations of students who have used the many editions of this text. They have taught us at least as much as we have taught them, and their questions and suggestions continue to improve the text and supplementary materials. Finally, we need to thank instructors from across the country who are continually sharing their knowledge and experience with us through market feedback and symposia. The feedback we ­received shaped this edition. All of these people took time to share their ideas and opinions to help us build a better edition of Biology for the next generation of introductory biology students, and they have our heartfelt thanks.

Reviewers for Biology, 13th edition Beth Cliffel Triton College David Cox Lincoln Land Community College Sarah Cuccinello University of Tampa Stella Doyungan Texas A&M University–Corpus Christi Sherry L. Harrel Eastern Kentucky University Christopher Ivey California State University, Chico Lori Kayes Oregon State University Nathan Lanning California State University, Los Angeles Karen Neal Reynolds Community College Bruce Stallsmith University of Alabama Shalini Upadhyaya Reynolds Community College Tom Warren Snead State Community College Jennifer Whitt Texas A&M University–Corpus Christi

Preparing Students for the Future Developing Critical Thinking with the Help of . . . Scientific Thinking Figures

Data Analysis Questions

Key illustrations in every chapter highlight how the frontiers of knowledge are pushed forward by a combination of hypothesis and experimentation. These figures begin with a hypothesis, then show how it makes explicit predictions, tests these by experiment, and finally demonstrates what conclusions can be drawn, and where this leads. Scientific Thinking figures provide a consistent framework to guide the student in the logic of scientific inquiry. Each illustration concludes with open-ended questions to promote scientific inquiry.

It’s not enough that students learn concepts and memorize scientific facts; a biologist needs to analyze data and apply that knowledge. Data Analysis questions inserted throughout the text challenge students to analyze data and interpret experimental results, which shows a deeper level of understanding.

Inquiry Questions

These questions challenge students to think about and engage in what they are reading at a more sophisticated level.

SCIENTIFIC THINKING Hypothesis: The plasma membrane is fluid, not rigid. Prediction: If the membrane is fluid, membrane proteins may diffuse laterally. Test: Fuse mouse and human cells, then observe the distribution of membrane proteins over time by labeling specific mouse and human proteins.

30 28 26 open habitat shaded forest

24

Human cell

Mouse cell

Body Temperature (°C)

32

24

26 28 30 Air Temperature (°C)

32

Figure 54.3 Behavioral adaptation. In open habitats, the Fuse cells

Intermixed membrane proteins

Allow time for mixing to occur Result: Over time, hybrid cells show increasingly intermixed proteins. Conclusion: At least some membrane proteins can diffuse laterally in the membrane. Further Experiments: Can you think of any other explanation for these observations? What if newly synthesized proteins were inserted into the membrane during the experiment? How could you use this basic experimental design to rule out this or other possible explanations?

Puerto Rican crested lizard, Anolis cristatellus, maintains a relatively constant temperature by seeking out and basking in patches of sunlight; as a result, it can maintain a relatively high temperature even when the air is cool. In contrast, in shaded forests, this behavior is not possible, and the lizard’s body temperature conforms to that of its surroundings.

(inset) Jonathan Losos

?

Inquiry question When given the opportunity, lizards regulate their body temperature to maintain a temperature optimal for physiological functioning. Would lizards in open habitats exhibit different escape behaviors from those of lizards in shaded forest?

Data analysis Can the slope of the line tell us something about the behavior of the lizard?

Figure 5.6 Test of membrane fluidity.

xv

Visual Maps

Visual Outline

Every chapter contains a list of chapter contents but now also contains a Visual Outline that shows topics in relation to each other. The Visual Outlines differ from “concept maps” in that there is a hierarchical aspect because they present a conceptual framework for the chapter. In looking at the Visual Outline before reading a chapter, students will be familiar with key terminology and how key topics generally relate to each other. Images are placed in the Visual Outline as a graphic connection to figures in the chapter.

There are two new but related features in Biology, 13th edition that help students build a conceptual framework into which they can insert new knowledge. Every chapter contains a Visual Outline at the beginning of the chapter and a more detailed Visual Summary at the end of the chapter. These complementary features serve as conceptual bookends to help students place chapter topics into a conceptual framework rather than a linear list. When topics are placed in the context of a map, students see relationships and connections between topics that are not apparent in the standard chapter outline and chapter review formats.

Fluid mosaic model

Cellular membranes

mediate

Transport 3 types

Consists of Passive transport Phospholipid bilayer

moves moves down up

Active transport

Bulk transport

Concentration gradient

3 types

Transmembrane proteins

Simple diffusion

Cytoskeleton

Osmosis

Cell surface markers

Facilitated diffusion

types Requires energy

Endocytosis Exocytosis

ATP

Dr. Edwin P. Ewing, Jr./CDC

Visual Summary

As an extension of the Visual Outline, a Visual Summary is presented at the end of all chapters following the more traditional textual summary. Like the chapter opening feature, the Visual Summary presents the chapter topics in relation to each other and

xvi Preparing Students for the Future

expands on the Visual Outline with supporting details of the key topics. The Visual Summary doesn’t contain all material in the chapter, nor does it indicate all connections between topics, but presents a conceptual scaffold. Students should be encouraged to build upon the scaffold with additional information from the chapter.

Fluid mosaic model

Consists of

Cellular membranes

mediate

Transport 3 types

Glycerol, 2 fatty acids, phosphate made of

Forms spontaneously

Phospholipid bilayer Transmembrane proteins

Fluid include

Passive transport 3 types

Transporters Enzymes

Cytoskeleton

Receptors Cell surface markers Function Cell recognition

types

Movement of water

Facilitated diffusion

Adhesion proteins

Selective

Glycolipids

Concentration difference

Osmosis

is

Glycoproteins

Concentration gradient defined as

Simple diffusion

Identity markers

Cytoskeleton attachment

move down

Carrier proteins uses Channel proteins

move up

Active transport

Bulk transport types

example Sodiumrequires potassium pump

Movement into the cell

Endocytosis is specific

Energy Carrier proteins

Receptormediated endocytosis

Exocytosis

Movement out of the cell

Preparing Students for the Future xvii

Strengthen Problem-Solving Skills with Connect® Detailed Feedback in Connect®

Learning is a process of iterative development, of making mistakes, reflecting, and adjusting over time. The question and test banks in Connect® for Biology, 13th edition, are more than direct assessments; they are self-contained learning experiences that systematically build student learning over time. For many students, choosing the right answer is not necessarily based on applying content correctly; it is more a matter of increasing their statistical odds of guessing. A major fault with this approach is that students don’t learn how to process the questions correctly, mostly because they are repeating and reinforcing their mistakes rather than reflecting and learning from them. To help students develop problemsolving skills, all higher-level Blooms questions in Connect are supported with hints, to help students focus on important information for answering the questions, and detailed feedback that walks students through the problem-solving process, using Socratic questions in a decision-tree-style framework to

xviii Preparing Students for the Future

scaffold learning, where each step models and reinforces the learning process. The feedback for each higher-level Blooms question (Apply, Analyze, Evaluate) follows a similar process: Clarify Question, Gather Content, Choose Answer, Reflect on Process.

Unpacking the Concepts

We’ve taken problem solving a step further. In each chapter, three to five higher-level Blooms questions in the question and test banks are broken out by the steps of the detailed feedback. Rather than leaving it up to the student to work through the detailed feedback, a second version of the question is presented in a stepwise format. Following the problem-solving steps, students need to answer questions about earlier steps, such as “What is the key concept addressed by the question?” before proceeding to answer the question. Found under the Coursewide Content section, a professor can choose which version of the question to include in the assignment based on the problem-solving skills of the students.

Data and Graphing Interactives To help students develop analytical skills, Connect® for Biology, 13th edition, is enhanced with Data and Graphing Interactives also found under the Coursewide Content. Students are

presented with a scientific problem and the opportunity to manipulate variables in the interactive, producing different results. A series of questions follows the activity to assess whether the student understands and is able to interpret the data and results.

McGraw Hill

Virtual Labs and Lab Simulations While the biological sciences are hands-on disciplines, instructors are now often being asked to deliver some of their lab components online, as full online replacements, supplements to prepare for in-person labs, or make-up labs. These simulations help each student learn the practical and conceptual skills needed, then check for understanding and

provide feedback. With adaptive pre-lab assignment, found under Adaptive Learning Assignment, and post-lab assessment available under Coursewide Content, instructors can customize each assignment. From the instructor’s perspective, these simulations may be used in the lecture environment to help students visualize complex scientific processes, such as DNA technology or Gram staining, while at the same time providing a valuable connection between the lecture and lab environments.

Preparing Students for the Future xix

Additional Assets in Connect® Coursewide Content

There are book-specific question and test banks in Connect® for Biology, 13th edition, but there are also additional assets under the Coursewide Content section. In addition to the Unpacking the Concepts and Data and Graphing Interactives mentioned earlier, this dropdown menu contains: Relevancy Modules, Quantitative Reasoning Questions, Virtual Labs Questions, BioNow Video ­Activities, and Biology NewsFlash Exercises.

SmartBook 2.0

Connect’s SmartBook 2.0 provides an adaptive learning experience that combines eBook reading for comprehension and assessments that test understanding. Learning resources are also available at key points to further aid understanding. The reading experience and assessments adapt to individual student learning. This is an environment that develops self-awareness through meaningful, immediate feedback that improves student success.

Prep for Majors Biology

Connect’s Prep is another adaptive learning experience. It is intended for use at the start of the majors biology course to get students up to speed on prerequisite material such as basic math skills, graphing, and statistics as well as introductory biology topics in chemistry and cell biology. An additional module, Fundamentals of Student Success, help students prepare for their college academic experience. Assessments determine a student’s prerequisite knowledge and learning resources help to fill in gaps in knowledge.

written communication skills and conceptual understanding. As an instructor you can assign, monitor, grade, and provide feedback on writing more efficiently and effectively.

ReadAnywhere

Read or study when it’s convenient for you with McGraw Hill’s free ReadAnywhere app. Available for iOS or Android smartphones or tablets, ReadAnywhere gives users access to McGraw Hill tools, including the eBook and SmartBook 2.0 or Adaptive Learning Assignments in Connect. Take notes, highlight, and complete ­assignments offline–all of your work will sync when you open the app with WiFi access. Log in with your McGraw Hill Connect username and password to start learning–anytime, anywhere!

OLC-Aligned Courses

Implementing High-Quality Online Instruction and Assessment through Preconfigured Courseware In consultation with the Online Learning Consortium (OLC) and our certified Faculty Consultants, McGraw Hill has created preconfigured courseware using OLC’s quality scorecard to align with best practices in online course delivery. This turnkey courseware contains a combination of formative assessments, summative assessments, homework, and application activities, and can easily be customized to meet an individual’s needs and course outcomes. For more information, visit https://www.mheducation .com/highered/olc.

Tegrity: Lectures 24/7

Remote Proctoring & Browser-Locking Capabilities

New remote proctoring and browser-locking capabilities, hosted by Proctorio within Connect®, provide control of the assessment environment by enabling security options and verifying the identity of the student. Seamlessly integrated within Connect, these services allow instructors to control students’ assessment experience by restricting browser activity, recording students’ activity, and verifying that students are doing their own work. Instant and detailed reporting gives instructors an at-a-glance view of potential academic integrity concerns, thereby avoiding personal bias and supporting evidence-based claims.

Writing Assignments

Available within McGraw Hill Connect®, the Writing Assignment tool delivers a learning experience to help students improve their

xx Preparing Students for the Future

Tegrity in Connect is a tool that makes class time available 24/7 by automatically capturing every lecture. With a simple one-click start-and-stop process, you capture all computer screens and corresponding audio in a format that is easy to search, frame by frame. Students can replay any part of any class with easy-to-use, browser-based viewing on a PC, Mac, tablet, or other mobile device. Educators know that the more students can see, hear, and experience class resources, the better they learn. In fact, studies prove it. Tegrity’s unique search feature helps students efficiently find what they need, when they need it, across an entire semester of class recordings. Help turn your students’ study time into learning moments immediately supported by your lecture. With Tegrity, you also increase intent listening and class participation by easing students’ concerns about note-taking. Using Tegrity in Connect will make it more likely you will see students’ faces, not the tops of their heads.

Test Builder in Connect

Available within Connect, Test Builder is a cloud-based tool that enables instructors to format tests that can be printed, administered within a Learning Management System, or exported as a Word document of the test bank. Test Builder offers a modern, streamlined interface for easy content configuration that matches course needs, without requiring a download. Test Builder allows you to: ■■

access all test bank content from a particular title.

■■

easily pinpoint the most relevant content through robust filtering options.

■■

manipulate the order of questions or scramble questions and/ or answers.

■■

pin questions to a specific location within a test.

■■

determine your preferred treatment of algorithmic questions.

■■

choose the layout and spacing.

■■

add instructions and configure default settings.

Test Builder provides a secure interface for better protection of content and allows for just-in-time updates to flow directly into assessments.

Create

Your Book, Your Way McGraw Hill’s Content Collections Powered by Create® is a selfservice website that enables instructors to create custom course materials—print and eBooks—by drawing upon McGraw Hill’s comprehensive, cross-disciplinary content. Choose what you want from our high-quality textbooks, articles, and cases. Combine it with your own content quickly and easily, and tap into other rights-secured, third-party content such as readings, cases, and articles. Content can be arranged in a way that makes the most sense for your course and you can include the course name and information as well. Choose the best format for your course: color print, black-and-white print, or eBook. The eBook can be included in your Connect course and is available on the free ReadAnywhere app for smartphone or tablet access as well. When you are finished customizing, you will receive a free digital copy to review in just minutes! Visit McGraw Hill Create®—www.mcgrawhillcreate.com—today and begin building!

Preparing Students for the Future xxi

Instructors: Student Success Starts with You Tools to enhance your unique voice Want to build your own course? No problem. Prefer to use an OLC-aligned, prebuilt course? Easy. Want to make changes throughout the semester? Sure. And you’ll save time with Connect’s auto-grading too.

65% Less Time Grading

Study made personal Incorporate adaptive study resources like SmartBook® 2.0 into your course and help your students be better prepared in less time. Learn more about the powerful personalized learning experience available in SmartBook 2.0 at www.mheducation.com/highered/connect/smartbook Laptop: McGraw Hill; Woman/dog: George Doyle/Getty Images

Affordable solutions, added value

Solutions for your challenges

Make technology work for you with LMS integration for single sign-on access, mobile access to the digital textbook, and reports to quickly show you how each of your students is doing. And with our Inclusive Access program you can provide all these tools at a discount to your students. Ask your McGraw Hill representative for more information.

A product isn’t a solution. Real solutions are affordable, reliable, and come with training and ongoing support when you need it and how you want it. Visit www .supportateverystep.com for videos and resources both you and your students can use throughout the semester.

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Students: Get Learning that Fits You Effective tools for efficient studying Connect is designed to help you be more productive with simple, flexible, intuitive tools that maximize your study time and meet your individual learning needs. Get learning that works for you with Connect.

Study anytime, anywhere Download the free ReadAnywhere app and access your online eBook, SmartBook 2.0, or Adaptive Learning Assignments when it’s convenient, even if you’re offline. And since the app automatically syncs with your Connect account, all of your work is available every time you open it. Find out more at www.mheducation.com/readanywhere

“I really liked this app—it made it easy to study when you don't have your textbook in front of you.” - Jordan Cunningham, Eastern Washington University

Everything you need in one place Your Connect course has everything you need—whether reading on your digital eBook or completing assignments for class, Connect makes it easy to get your work done.

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PartCHAPTER VIII Genetic and Molecular Biology Part I The Molecular Basis of Life

1

The Science of Biology Chapter Contents 1.1

The Science of Life

1.2

The Nature of Science

1.3

An Example of Scientific Inquiry: Darwin and Evolution

1.4

Core Concepts in Biology

Soames Summerhays/Natural Visions

Introduction

Visual Outline Methods of science

Biology

Living systems de a fin m for by ed

use Observation

Reasoning

Hierarchy

unified by

Shared characteristics

O H

C N

Core concepts include

Chemical and physical laws

Structure determines function

Energy transformations

Information transactions

Evolution

(Nerve cell): SPL/Science Source; (Ducks and geese on pond): George Ostertaga/gefotostock/Alamy Stock Photo

You are about to embark on a journey—a journey of discovery about the nature of life. More than 180 years ago, a young English naturalist named Charles Darwin set sail on a similar journey on board H.M.S. Beagle; a replica of this ship is pictured here. What Darwin learned on his five-year voyage led directly to his development of the theory of evolution by natural selection, a theory that has become the core of the science of biology. Darwin’s voyage seems a fitting place to begin our exploration of biology—the scientific study of living organisms and how they have evolved. Before we begin, however, let’s take a moment to think about what biology is and why it’s important.

1.1 The

introductory chapter, we examine the nature of biology and the foundations of science in general to put into context the information presented in the rest of the text.

Science of Life

Biology unifies much of natural science

Learning Outcomes 1. Compare biology to other natural sciences. 2. Describe the characteristics of living systems. 3. Characterize the hierarchical organization of living systems.

This is the most exciting time to be studying biology in the history of the field. The amount of information available about the natural world has exploded in the last 50 years, since the construction of the first recombinant DNA molecule. We are now in a position to ask and answer questions that previously were only dreamed of. The 21st century began with the completion of the sequence of the human genome. The largest single project in the history of biology took about 20 years. Yet less than 20 years later, we can sequence an entire genome in a matter of days. This flood of sequence data and genomic analysis are altering the landscape of biology. These and other discoveries are also moving into the clinic as never before, with new tools for diagnostics and treatment. With robotics, next-generation DNA sequencing technologies, advanced imaging, and analytical techniques, we have tools available that were formerly the stuff of science fiction. In this text, we attempt to draw a contemporary picture of the science of biology, as well as provide some history and experimental perspective on this exciting time in the discipline. In this

The study of biology is a point of convergence for the information and tools from all of the natural sciences. Biological systems are the most complex chemical systems on Earth, and their many functions are both determined and constrained by the principles of chemistry and physics. Put another way, no new laws of nature can be gleaned from the study of biology—but that study does illuminate and illustrate the workings of those natural laws. The intricate chemical workings of cells can be understood using the tools and principles of chemistry. And every level of biological organization is governed by the nature of energy transactions first studied by thermodynamics. Biological systems do not represent any new forms of matter, and yet they are the most complex organization of matter known. The complexity of living systems is made possible by a constant source of energy—the Sun. The conversion of this radiant energy into organic molecules by photosynthesis is one of the most beautiful and complex reactions known in chemistry and physics. The way we do science is changing to grapple with increasingly difficult modern problems. Science is becoming more interdisciplinary, combining the expertise from a variety of traditional disciplines and emerging fields such as nanotechnology. Biology is at the heart of this multidisciplinary approach because biological problems often require many different approaches to arrive at solutions.

CELLULAR LEVEL Atoms

Molecule

Macromolecule

Organelle

Cell

Tissue

Organ

O C H N O H N C O 0.2 μm

2 part I The Molecular Basis of Life

100 μm

Life defies simple definition

■■

In its broadest sense, biology is the study of living things—the ­science of life. Living things come in an astounding variety of shapes and forms, and biologists study life in many different ways. They live with gorillas, collect fossils, and listen to whales. They read the messages encoded in the long molecules of heredity and count how many times a hummingbird’s wings beat each second. What makes something “alive”? Anyone could deduce that a galloping horse is alive and a car is not, but why? We cannot say, “If it moves, it’s alive,” because a car can move, and gelatin can wiggle in a bowl. They certainly are not alive. Although we cannot define life with a single simple sentence, we can come up with a series of seven characteristics shared by living systems: ■■

■■

■■

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Cellular organization. All organisms consist of one or more cells. Often too tiny to see, cells carry out the basic activities of living. Each cell is bounded by a membrane that separates it from its surroundings. Ordered complexity. All living things are both complex and highly ordered. Your body is composed of many different kinds of cells, each containing many complex molecular structures. Many nonliving things may also be complex, but they do not exhibit this degree of ordered complexity. Sensitivity. All organisms respond to stimuli. Plants grow toward a source of light, and the pupils of your eyes dilate when you walk into a dark room. Growth, development, and reproduction. All organisms are capable of growing and reproducing, and they all possess hereditary molecules that are passed to their offspring, ensuring that the offspring are of the same species.

Organism

■■

Living systems show hierarchical organization The organization of the biological world is hierarchical—that is, each level builds on the level below it: 1. The cellular level. At the cellular level (figure 1.1), atoms, the fundamental elements of matter, are

Figure 1.1 Hierarchical organization of living systems. Life forms a hierarchy of organization from atoms to complex multicellular organisms. Atoms are joined together to form molecules, which are assembled into more complex structures such as organelles. These in turn form subsystems that provide different functions. Cells can be organized into tissues, then into organs and organ systems such as the goose’s nervous system pictured. This organization then extends beyond individual organisms to populations, communities, ecosystems, and finally the biosphere. (Organelle): Keith R. Porter/Science Source; (Cell): SPL/Science Source; (Tissue): Ed Reschke; (Organism): Russell Illig/Getty Images; (Population): George Ostertaga/ gefotostock/Alamy Stock Photo; (Species): Sander Meertins/iStock/Getty Images; Pictureguy/Shutterstock; (Community): Ryan McGinnis/Alamy Stock Photo; (Ecosystem): Robert and Jean Pollock; (Biosphere): Goddard Space Flight Center/NASA

POPULATIONAL LEVEL

ORGANISMAL LEVEL Organ system

■■

Energy utilization. All organisms take in energy and use it to perform many kinds of work. Every muscle in your body is powered with energy you obtain from your diet. Homeostasis. All organisms maintain relatively constant internal conditions that are different from their environment, a process called homeostasis. For example, your body temperature remains stable despite changes in outside temperatures. Evolutionary adaptation. All organisms interact with other organisms and the nonliving environment in ways that influence their survival, and as a consequence, organisms evolve adaptations to their environments.

Population

Species

Community

Ecosystem

Biosphere

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joined together into clusters called molecules. Complex biological molecules are assembled into tiny structures called organelles within membranebounded units we call cells. The cell is the basic unit of life. Many i­ ndependent organisms are composed only of single cells. Bacteria are single cells, for example. All animals and plants, as well as most fungi and algae, are multicellular—composed of more than one cell. 2. The organismal level. Cells in complex multicellular organisms exhibit three levels of organization. The most basic level is that of tissues, which are groups of similar cells that act as a functional unit. Tissues, in turn, are grouped into organs—body structures composed of several different tissues that act as a structural and ­f unctional unit. Your brain is an organ composed of nerve cells and a variety of associated tissues that form protective coverings and contribute blood. At the third level of organization, organs are grouped into organ systems. The nervous system, for example, consists of sensory organs, the brain and spinal cord, and neurons that convey signals. 3. The populational level. Individual organisms can be categorized into several hierarchical levels within the living world. The most basic of these is the population—a group of organisms of the same species living in the same place. All populations of a particular kind of organism together form a species, its members similar in appearance and able to interbreed. At a higher level of biological organization, a biological community consists of all the populations of different species living together in one place. 4. The ecosystem level. At the highest tier of biological organization, populations of organisms interact with each other and their physical environment. Together populations and their environment constitute an ecological system, or ecosystem. For example, the biological community of a mountain meadow interacts with the soil, water, and atmo­ sphere of a mountain ecosystem in many important ways. 5. The biosphere. The entire planet can be thought of as an ecosystem that we call the biosphere. As you move up this hierarchy, the many interactions occurring at lower levels can produce novel properties. These so-called emergent properties may not be predictable. Examining individ­ ual cells, for example, gives little hint about the whole animal. Many weather phenomena, such as hurricanes, are actually emergent properties of many interacting meteorological variables. It is because the living world exhibits many emergent properties that it is difficult to define “life.” This description of the common features and organization of living systems provides an introduction for our exploration of biology. Before we continue, we will consider the broader question of the nature of science itself.

4 part I The Molecular Basis of Life

Learning Outcomes Review 1.1

Biology as a science brings together other natural sciences, such as chemistry and physics, to study living systems. Life does not have a simple definition, but living systems share a number of properties that together describe life. Living systems can be organized hierarchically, from the cellular level to the entire biosphere, with emergent properties that may exceed the sum of the parts. ■■

Can you study biology without studying other sciences?

1.2 The

Nature of Science

Learning Outcomes 1. Compare the different types of reasoning used by biologists. 2. Demonstrate how to formulate and test a hypothesis.

Much like life itself, the nature of science defies simple description. For many years scientists have written about the “scientific method” as though there is a single way of doing science. This oversimplification has contributed to confusion on the part of nonscientists about the nature of science. At its core, science is concerned with developing an increasingly accurate understanding of the world around us using observation and reasoning. To begin with, we assume that natural forces acting now have always acted, that the fundamental nature of the universe has not changed since its in­ception, and that it is not changing now. A number of complementary approaches allow understanding of natural phenomena—there is no one “scientific method.” Scientists also attempt to be as objective as possible in the interpretation of the data and observations they have collected. Because scientists themselves are human, this is not completely possible, but because science is a collective endeavor subject to scrutiny, it is self-correcting. One person’s results are verified by others, and if the results cannot be repeated, they are rejected.

Much of science is descriptive The classic vision of the scientific method is that observations lead to hypotheses that in turn make experimentally testable predictions. In this way, we dispassionately evaluate new ideas to arrive at an increasingly accurate view of nature. We discuss this way of doing science later in this section, but it is important to understand that much of science is purely descriptive: in order to understand anything, the first step is to describe it completely. Much of biology is concerned with arriving at an increasingly accurate description of nature. The study of biodiversity is an example of descriptive science that has implications for other aspects of biology in addition to societal implications. Efforts are currently under way to classify all life on Earth. This ambitious project is purely descriptive, but it will lead to a much greater understanding of biodiversity as well as the effect our species has on biodiversity.

Sunlight at midday

Height of obelisk

Well

Light rays parallel

a Distance between cities = 800 km Length of shadow

a

Figure 1.2 Deductive reasoning: How Eratosthenes estimated the circumference of the Earth using deductive reasoning. 1. On a day when sunlight shone straight down a deep well at

Syene in Egypt, Eratosthenes measured the length of the shadow cast by a tall obelisk in the city of Alexandria, about 800 kilometers (km) away. 2. The shadow’s length and the obelisk’s height formed two sides of a triangle. Using the recently developed principles of Euclidean geometry, Eratosthenes calculated the angle, a, to be 7° and 12´, exactly 1⁄50 of a circle (360°). 3. If angle a is 1⁄50 of a circle, then the distance between the obelisk (in Alexandria) and the well (in Syene) must be equal to 1⁄50 the circumference of the Earth. 4. Eratosthenes had heard that it was a 50-day camel trip from Alexandria to Syene. Assuming a camel travels about 18.5 km per day, he estimated the distance between obelisk and well as 925 km (using different units of measure, of course). 5. Eratosthenes thus deduced the circumference of the Earth to be 50 × 925 = 46,250 km. Modern measurements put the distance from the well to the obelisk at just over 800 km. Using this distance Eratosthenes’s value would have been 50 × 800 = 40,000 km. The actual circumference is 40,075 km.

One of the most important accomplishments of molecular biology at the dawn of the 21st century was the completion of the sequence of the human genome. Many new hypotheses about human biology will be generated by this knowledge, and many experiments will be needed to test these hypotheses, but the determination of the sequence itself was descriptive science.

Science uses both deductive and inductive reasoning The study of logic recognizes two opposite ways of arriving at logical conclusions: deductive and inductive reasoning. Science makes use of both of these methods, although induction is the ­primary way of reasoning in hypothesis-driven science.

Deductive reasoning Deductive reasoning applies general principles to predict specific results. More than 2200 years ago, the Greek scientist ­Eratosthenes used Euclidean geometry and deductive reasoning to accurately estimate the circumference of the Earth (­figure  1.2). Deductive reasoning is the reasoning of mathematics and ­philosophy, and it is used to test the validity of general ideas in all branches of knowledge. For example, if all mammals by definition have hair, and you find an animal that does not have hair, then you may conclude that this animal is not a mammal. A biologist uses deductive reasoning to infer the species of a specimen from its characteristics.

Inductive reasoning In inductive reasoning, the logic flows in the opposite direction, from the specific to the general. Inductive reasoning uses specific observations to construct general scientific principles. For example, if poodles have hair, and terriers have hair, and every dog that you observe has hair, then you may conclude that all dogs have

hair. Inductive reasoning leads to generalizations that can then be tested. Inductive reasoning first became important to science in the 1600s in Europe, when Francis Bacon, Isaac Newton, and others began to use the results of particular experiments to infer general principles about how the world operates. An example from modern biology is the role of specific genes in development. Studies in the fruit fly, Drosophila melanogaster, identified genes that could cause dramatic changes in developmental fate, such as a leg appearing in the place of an antenna. These genes have since been found in essentially all multicellular animals analyzed. This led to the general idea that the same kind of genes control developmental fate in all animals.

Hypothesis-driven science makes and tests predictions Scientists establish which general principles are true from among the many that might be true through the process of systematically testing alternative proposals. If these proposals prove inconsistent with experimental observations, they are rejected as untrue. Figure 1.3 illustrates the process. After making careful observations, scientists construct a hypothesis, which is a suggested explanation that accounts for those observations. A hypothesis is a proposition that might be true. Those hypotheses that have not yet been disproved are retained. They are useful because they fit the known facts, but they are always subject to future rejection if, in the light of new information, they are found to be incorrect. This is usually an ongoing process with a hypothesis changing and being refined with new data. For instance, geneticists George Beadle and Edward Tatum studied the nature of genetic information to arrive at their “one-gene/one-enzyme” hypothesis (see chapter 15). This hypothesis states that a gene represents the genetic information necessary to make a single enzyme. As investigators learned more about the molecular nature of genetic

1

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Problem

Collect observations Induction Generate hypotheses Deduction Modify hypothesis or generate new hypothesis

Generate testable predictions

Experimental test of predictions

Falsification

Reject hypothesis

Hypothesis supported

Replication and new tests

Eventual falsification

Hypothesis supported

Modify hypothesis or generate new hypothesis

Figure 1.3 Testing hypotheses with experiments. This provides a general flowchart for testing hypotheses by experimentation. A problem of interest is identified and observations are collected. Inductive reasoning leads to development of one or more potential explanations (hypotheses). Experimental results will either support or falsify a hypothesis. A hypothesis that is supported is subject to further replication and testing, leading to either eventual rejection or further support. Falsified hypotheses can be modified or rejected in favor of a new hypothesis.

information, the hypothesis was refined to “one gene/one polypeptide” because enzymes can be made up of more than one polypeptide. With still more information about the nature of genetic information, other investigators found that a single gene can specify more than one polypeptide, and the hypothesis was refined again.

Testing hypotheses We call the test of a hypothesis an experiment. Suppose you enter a dark room. To understand why it is dark, you propose several hypotheses. The first might be, “There is no light in the room because the light switch is turned off.” An alternative hypothesis might be, “There is no light in the room because the lightbulb is burned out.” And yet another hypothesis might be, “I am going blind.” To evaluate these hypotheses, you would conduct an experiment designed to eliminate one or more of the hypotheses. For example, you might test your hypotheses by flipping the light switch. If you do so and the room is still dark, you have disproved the first hypothesis: something other than the setting of the light switch must be the reason for the darkness. Note that a test such as this does not prove that any of the other hypotheses are true; it merely demonstrates that the one being tested is not. A 6 part I The Molecular Basis of Life

successful experiment is one in which one or more of the alternative hypotheses is demonstrated to be inconsistent with the results and is thus rejected. As you proceed through this text, you will encounter many hypotheses that have withstood the test of experiment. Many will continue to do so; others will be revised as new observations are made by biologists. Biology, like all science, is in a constant state of change, with new ideas appearing and replacing or refining old ones.

Establishing controls Often scientists are interested in learning about processes that are influenced by many factors, or variables. To evaluate alternative hypotheses, we keep the variable of interest constant in a control group, and alter it in the experimental treatment. This allows us to isolate the effects of a single variable in our experiment, so any difference in the outcome must result from the influence of this variable. Much of the challenge of experimental science lies in designing controls that isolate a particular variable from other factors that might influence a process.

Using predictions For a scientific hypothesis to be successful, it must not only explain and unify observations, but also must make testable predictions. When proposing a hypothesis, you need to keep in mind that it may explain everything you have observed, but also be completely wrong. As Thomas Huxley, a contemporary and supporter of Charles Darwin, put it: “The great tragedy of science, the slaying of a beautiful theory by an ugly fact.” An example from the early history of microbiology involves the observation that nutrient broth left exposed to air becomes contaminated. Two hypotheses were proposed to explain this observation: spontaneous generation and the germ hypothesis. Spontaneous generation held that there was an inherent property in organic molecules that could lead to the spontaneous generation of life. The germ hypothesis proposed that preexisting microorganisms that were present in the air could contaminate the nutrient broth. These competing hypotheses were tested by a number of experiments that involved filtering air and boiling the broth to kill any contaminating germs. The definitive experiment was performed by Louis Pasteur, who constructed flasks with curved necks that could be exposed to air, but that would trap any contaminating germs. When such flasks were boiled to sterilize them, they remained sterile, but if the curved neck was broken off, they became contaminated (figure 1.4). This result provides a clear distinction between the two hypotheses: the germ theory predicts the results shown, while spontaneous generation predicts growth in either case. These results, then, support the germ hypothesis and cause us to reject spontaneous generation.

Reductionism breaks larger systems into their component parts Scientists use the philosophical approach of r­eductionism to understand a complex system by reducing it to its working parts. Reductionism has been the general approach of biochemistry, which has been enormously successful at unraveling the complexity of cellular metabolism by concentrating on individual pathways

SCIENTIFIC THINKING Question: What is the source of contamination that occurs in a flask of nutrient broth left exposed to the air? Germ Hypothesis: Preexisting microorganisms present in the air contaminate nutrient broth. Prediction: Sterilized broth will remain sterile if microorganisms are prevented from entering flask. Spontaneous Generation Hypothesis: Living organisms will spontaneously generate from nonliving organic molecules in broth. Prediction: Organisms will spontaneously generate from organic molecules in broth after sterilization. Test: Use swan-necked flasks to prevent entry of microorganisms. To ensure that broth can still support life, break swan-neck after sterilization. Broken neck of flask

Flask is sterilized by boiling the broth.

Unbroken flask remains sterile.

Broken flask becomes contaminated after exposure to germ-laden air.

Result: No growth occurs in sterile swan-necked flasks. When the neck is broken off, and the broth is exposed to air, growth occurs. Conclusion: Growth in broth is of preexisting microorganisms.

Figure 1.4 Experiment to test spontaneous generation versus germ hypothesis. and specific enzymes. By analyzing all of the pathways and their components, scientists now have an overall picture of the metabolism of cells. Reductionism has limits when applied to living systems, however—one of which is that enzymes do not always behave exactly the same in isolation as they do in their normal cellular context. A larger problem is that the complex interworking of many interconnected functions leads to emergent properties that cannot be predicted based on the workings of the parts. For example, ribosomes are the cellular factories that synthesize proteins, but this function could not be predicted based on analysis of the individual proteins and RNA that make up the structure. On a higher level, understanding the physiology of a single Canada goose would not lead to predictions about flocking behavior. The emerging field of systems biology uses mathematical and computational models to deal with the whole as well as understanding the interacting parts.

Biologists construct models to explain living systems Biologists construct models in many different ways for a variety of uses. Geneticists construct models of interacting networks of proteins that control gene expression, often even drawing cartoon figures to represent that which we cannot see. Population biologists build models of how evolutionary change occurs. Cell biologists build models of signal transduction pathways and the events leading from an external signal to internal events. Structural

biologists build actual models of the structure of proteins and macromolecular complexes in cells. Models provide a way to organize how we think about a problem. Models can also get us closer to the larger picture and away from the extreme reductionist approach. The working parts are provided by the reductionist analysis, but the model shows how they fit together. Often these models suggest other experiments that can be performed to refine or test the model. As researchers gain more knowledge about the actual flow of molecules in living systems, more sophisticated kinetic models can be used to apply information about isolated enzymes to their cellular context. In systems biology, this modeling is being applied on a large scale to regulatory networks during development, and even to modeling an entire bacterial cell.

The nature of scientific theories Scientists use the word theory in two main ways. The first meaning of theory is a proposed explanation for some natural phenomenon, often based on some general principle. Thus, we speak of the principle first proposed by Newton as the “theory of gravity.” Such theories often bring together concepts that were previously thought to be unrelated. The second meaning of theory is the body of interconnected concepts, supported by scientific reasoning and experimental evidence, that explains the facts in some area of study. Such a theory provides an indispensable framework for organizing a body of knowledge. For example, quantum theory in physics brings together a set of ideas about the nature of the universe, explains experimental facts, and serves as a guide to further questions and experiments. To a scientist, theories are the solid ground of science, expressing ideas of which we are most certain. In contrast, to the general public, the word theory usually implies the opposite—a lack of knowledge, or a guess. Not surprisingly, this difference often results in confusion. In this text, theory will always be used in its scientific sense, in reference to an accepted general principle or body of knowledge. Some critics outside of science attempt to discredit evolution by saying it is “just a theory.” The hypothesis that evolution has occurred, however, is an accepted scientific fact—it is supported by overwhelming evidence. Modern evolutionary theory is a complex body of ideas, the importance of which spreads far beyond explaining evolution. Its ramifications permeate all areas of biology, and it provides the conceptual framework that unifies biology as a science. Again, the key is how well a hypothesis fits the observations. Evolutionary theory fits the observations very well.

Research can be basic or applied In the past it was fashionable to speak of the “scientific method” as consisting of an orderly sequence of logical, either–or steps. Each step would reject one of two mutually incompatible alternatives, as though trial-and-error testing would inevitably lead a researcher through the maze of uncertainty to the ultimate scientific answer. If this were the case, a computer would make a good scientist. But science is not done this way. As the British philosopher Karl Popper has pointed out, successful scientists without exception design their experiments with a pretty fair idea of how the results are going to come out. They have

1

chapter  The Science of Biology 7

what Popper calls an “imaginative preconception” of what the truth might be. Because insight and imagination play such a large role in scientific progress, some scientists are better at science than others— just as Bruce Springsteen stands out among songwriters or Claude Monet stands out among Impressionist painters. Some scientists perform basic research, which is intended to extend the boundaries of what we know. These individuals typically work at universities, and their research is usually supported by grants from various agencies and foundations. The information generated by basic research contributes to the growing body of scientific knowledge, and it provides the scientific foundation utilized by applied research. Scientists who conduct applied research are often employed in some kind of industry. Their work may involve the manufacture of food additives, the creation of new drugs, or the testing of environmental quality. Research results are published in scientific journals, where the experiments and conclusions are reviewed by other scientists. This process of careful evaluation, called peer review, lies at the heart of modern science. It helps to ensure that faulty research or false claims are challenged and not accepted without examination. Recently, there have been problems raised with reproducibility in some areas of biology. That this is being examined indicates the self-reflective nature of science. With some idea of what science is and how it functions, we will consider a single example in detail. What better example than the development of one of the most important ideas in the history of science: Darwin’s theory of evolution by natural selection.

Learning Outcomes Review 1.2

Much of science is descriptive, amassing observations to gain an accurate view. Both deductive reasoning and inductive reasoning are used in science. Scientific hypotheses are suggested explanations for observed phenomena. Hypotheses need to make predictions that can be tested by controlled experiments. Theories are coherent explanations of observed data, but they may be modified by new information. ■■

How does a scientific theory differ from a hypothesis?

1.3 An

Example of Scientific Inquiry: Darwin and Evolution

Learning Outcomes 1. Examine Darwin’s theory of evolution by natural selection as a scientific theory. 2. Describe the evidence that supports the theory of evolution.

Darwin’s theory of evolution explains and describes how organisms on Earth have changed over time and acquired a diversity of new forms. This famous theory provides a good example of how a 8 part I The Molecular Basis of Life

Figure 1.5 Charles Darwin. This newly rediscovered photograph taken in 1881, the year before Darwin died, appears to be the last ever taken of the great biologist. Huntington Library/Superstock

scientist develops a hypothesis and how a scientific theory grows and wins acceptance. Charles Robert Darwin (1809–1882; figure 1.5) was an English naturalist who, after 30 years of study and observation, wrote one of the most famous and influential books of all time. This book, On the Origin of Species by Means of Natural S­ election, created a sensation when it was published, and the ideas Darwin expressed in it have played a central role in the development of human thought ever since.

The idea of evolution existed prior to Darwin In Darwin’s time, most people believed that the different kinds of organisms and their individual structures resulted from direct actions of a Creator (many people still believe this). Species were thought to have been specially created and to be unchangeable over the course of time. In contrast to these ideas, a number of earlier naturalists and philosophers had presented the view that living things must have changed during the history of life on Earth. That is, e­ volution has occurred, and living things are now different from how they began. Darwin’s contribution was a concept he called natural ­selection, which he proposed as a coherent, logical explanation for this ­process, and he brought his ideas to wide public attention.

Darwin observed differences in related organisms The story of Darwin and his theory begins in 1831, when he was 22 years old. He was part of a five-year navigational mapping expedition around the coasts of South America (­figure  1.6), aboard H.M.S. Beagle. During this long voyage, Darwin had the chance to study a wide variety of plants and animals on continents and islands and in distant seas. Darwin observed a number of phenomena that were of central importance to his reaching his ultimate conclusion. Repeatedly, Darwin saw that the characteristics of similar species varied somewhat from place to place. These geographical patterns suggested to him that lineages change gradually as species

British Isles

NORTH PAC I F I C OCEAN

NORTH AMERICA

Galápagos Islands

NORTH AT L A N T I C OCEAN

Valparaiso Society Islands Straits of Magellan Cape Horn

INDIAN O C E A N Keeling Islands Madagascar

St. Helena Rio de Janeiro

Montevideo Buenos Aires Port Desire Falkland Islands Tierra del Fuego

NORTH PAC I F I C OCEAN Philippine Islands

AFRICA

Ascension

Bahia

ASIA

Canary Islands

Cape Verde Islands SOUTH AMERICA

Marquesas

EUROPE

Western Isles

Mauritius Bourbon Island Cape of Good Hope

SOUTH AT L A N T I C OCEAN

Equator

AUSTRALIA

Friendly Islands

Sydney King George’s Sound

Hobart

New Zealand

Figure 1.6 The five-year voyage of H.M.S. Beagle. Most of the time was spent exploring the coasts and coastal islands of South America, such as the Galápagos Islands. Darwin’s studies of the animals of the Galápagos Islands played a key role in his eventual development of the concept of evolution by means of natural selection. migrate from one area to another. On the Galápagos Islands, 960 km (600 miles) off the coast of Ecuador, Darwin encountered a variety of different finches on the various islands. The 14 species, although related, differed slightly in appearance, particularly in their beaks (figure 1.7). Darwin thought it was reasonable to assume that all these birds had descended from a common ancestor arriving from the South American mainland several million years ago. Eating

Woodpecker Finch (Cactospiza pallida)

different foods on different islands, the finches’ beaks had changed during their descent—“descent with modification,” or evolution. (These finches are discussed in more detail in chapters 21 and 22.) In a more general sense, Darwin was struck by the fact that the plants and animals on these relatively young volcanic islands resembled those on the nearby coast of South America. If each one of these plants and animals had been created independently and simply placed on the Galápagos Islands, why didn’t they resemble

Large Ground Finch (Geospiza magnirostris)

Cactus Finch (Geospiza scandens)

Figure 1.7 Three Galápagos finches and what they eat. On the Galápagos Islands, Darwin observed 14 different species of finches differing mainly in their beaks and feeding habits. These three finches eat very different food items, and Darwin surmised that the different shapes of their bills represented evolutionary adaptations that improved their ability to eat the foods available in their specific habitats.

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the plants and animals of islands with similar climates—such as those off the coast of Africa, for example? Why did they resemble those of the adjacent South American coast instead?

Darwin proposed natural selection as a mechanism for evolution It is one thing to observe the results of evolution, but quite another to understand how it happens. Darwin’s great achievement lies in his ability to move beyond all the individual observations to formulate the hypothesis that evolution occurs because of natural selection.

Darwin and Malthus Of key importance to the development of Darwin’s insight was his study of Thomas Malthus’s An Essay on the Principle of Population (1798). In this book, Malthus stated that populations of plants and animals (including humans) tend to increase geometrically, while humans are able to increase their food supply only arithmetically. Put another way, population increases by a multiplying factor—for example, in the series 2, 6, 18, 54, the starting number is multiplied by 3. Food supply increases by an additive factor—for example, the series 2, 4, 6, 8 adds 2 to each starting number. Figure 1.8 shows the difference that these two types of relationships produce over time. geometric progression arithmetic progression

54

Because populations increase geometrically, virtually any kind of animal or plant, if it could reproduce unchecked, would cover the entire surface of the world surprisingly quickly. Instead, populations of species remain fairly constant year after year, because death limits population numbers. Sparked by Malthus’s ideas, Darwin saw that although every organism has the potential to produce more offspring than can survive, only a limited number actually do survive and produce further offspring. Combining this observation with what he had seen on the voyage of the Beagle, as well as with his own experiences in breeding domestic animals, Darwin made an important association: individuals possessing physical, behavioral, or other attributes that give them an advantage in their environment are more likely to survive and reproduce than those with less advantageous traits. By surviving, these individuals gain the opportunity to pass on their favorable characteristics to their offspring. As the frequency of these characteristics increases in the population, the nature of the population as a whole will gradually change. Darwin called this process selection.

Natural selection Darwin was thoroughly familiar with variation in domesticated animals, and he began On the Origin of Species with a detailed discussion of pigeon breeding. He knew that animal breeders selected certain varieties of pigeons and other animals, such as dogs, to produce certain characteristics, a process Darwin called artificial selection. Artificial selection often produces a great variation in traits. Domestic pigeon breeds, for example, show much greater variety than all of the wild species found throughout the world. Darwin thought that this type of change could occur in nature, too. Surely if pigeon breeders could foster variation by artificial selection, nature could do the same—a process Darwin called natural selection.

Darwin drafts his argument 18

6 2

4

6

8

Figure 1.8 Geometric and arithmetic progressions. A geometric progression increases by a constant factor (for example, the curve shown increases ×3 for each step), whereas an arithmetic progression increases by a constant difference (for example, the line shown increases +2 for each step). Malthus contended that the human growth curve was geometric, but the human food production curve was only arithmetic.

Data analysis What is the effect of reducing the constant factor for a geometric progression? How would this change the curve in the figure?

?

Inquiry question Might this effect be achieved with humans? How?

10 part I The Molecular Basis of Life

Darwin drafted the overall argument for evolution by natural selection in a preliminary manuscript in 1842. After showing the manuscript to a few of his closest scientific friends, however, Darwin put it in a drawer, and for 16 years turned to other research. No one knows for sure why Darwin did not publish his initial manuscript— it is very thorough and outlines his ideas in detail. The stimulus that finally brought Darwin’s hypothesis into print was an essay he received in 1858. A young English naturalist named Alfred Russel Wallace (1823–1913) sent the essay to Darwin from Indonesia; it concisely set forth the hypothesis of evolution by means of natural selection, a hypothesis Wallace had developed independently of Darwin. After receiving Wallace’s essay, friends of Darwin arranged for a joint presentation of their ideas at a seminar in London. Darwin then completed his own book, expanding the 1842 manuscript he had written so long ago, and submitted it for publication.

The predictions of natural selection have been tested More than 130 years have elapsed since Darwin’s death in 1882. During this period, the evidence supporting his theory has grown

Figure 1.9 Homology among vertebrate limbs. 

The forelimbs of these five vertebrates show the ways in which the relative proportions of the forelimb bones have changed in relation to the particular way of life of each organism.

Human

Cat

Bat

progressively stronger. We briefly explore some of this evidence here; in chapter 21, we will return to the theory of evolution by natural selection and examine the evidence in more detail.

The fossil record Darwin predicted that the fossil record would yield intermediate links between the great groups of organisms—for example, between fishes and the amphibians thought to have arisen from them, and between reptiles and birds. Furthermore, natural selection predicts the relative positions in time of such transitional­forms. We now know the fossil record to a degree that was unthinkable in the 19th century, and although truly “intermediate” organisms are hard to determine, paleontologists have found what appear to be transitional forms and found them at the predicted positions in time. Analysis of microscopic fossils extends the history of life on Earth to about 3.5 billion years ago (bya). The discovery of other fossils has supported Darwin’s predictions and has shed light on how organisms have, over this enormous time span, evolved from the simple to the complex. For vertebrate animals especially, the fossil record is rich and exhibits a graded series of changes in form, with the evolutionary sequence visible for all to see.

The age of the Earth Darwin’s theory predicted the Earth must be very old, but some physicists argued that the Earth was only 100 million years old. This bothered Darwin, because this did not seem to allow enough time for the evolution of all living things from a common ancestor. Using evidence obtained by studying the rates of radioactive decay, we now know that the physicists of Darwin’s time were very wrong: the Earth was formed about 4.5 bya.

The mechanism of heredity Darwin received some of his sharpest criticism in the area of heredity. At that time, no one had any concept of genes or how heredity works, so it was not possible for Darwin to explain completely how evolution occurs. Even though Gregor Mendel was performing his experiments with pea plants in Brünn, Austria (now Brno, the Czech Republic), during roughly the same period, genetics was established as a science only at the start of the 20th century. When scientists began to understand the laws of inheritance (discussed in chapters 12 and 13), this problem with Darwin’s theory vanished.

Porpoise

Horse

Comparative anatomy Comparative studies of animals have provided strong evidence for Darwin’s theory. In many different types of vertebrates, for example, the same bones are present, indicating their evolutionary past. Thus, the forelimbs shown in figure 1.9 are all constructed from the same basic array of bones, modified for different purposes. These bones are said to be homologous in the different vertebrates—that is, they have the same evolutionary origin, but they now differ in structure and function. They are contrasted with analogous structures, such as the wings of birds and butterflies, which have similar function but different evolutionary origins.

Molecular evidence Evolutionary patterns are also revealed at the molecular level. By comparing the genomes (that is, the sequences of all the genes) of different groups of animals or plants, we can more precisely specify the degree of relationship among the groups. A series of evolutionary changes over time should involve a continual accumulation of genetic changes in the DNA. This difference can be seen clearly in the protein hemoglobin (figure 1.10). Rhesus monkeys, which like humans are primates, have fewer differences from humans in the 146-amino-acid hemoglobin β chain than do more distantly related mammals, such as dogs. Nonmammalian vertebrates, such as birds and frogs, differ even more. This kind of analysis allows us to construct phylogenetic trees that provide a graphic representation of these evolutionary relationships. We will explore these ideas in much more detail in Part IV. For now we will conclude our introduction to biology by considering how we can use core concepts to organize our thinking and deal with the enormous amount of information that is modern biology.

Learning Outcomes Review 1.3

Darwin observed differences in related organisms and proposed the hypothesis of evolution by natural selection to explain these differences. The predictions generated by natural selection have been tested and continue to be tested by analysis of the fossil record, genetics, comparative anatomy, and even the DNA of living organisms. ■■

Does Darwin’s theory of evolution by natural selection explain the origin of life?

1

chapter  The Science of Biology 11

Human

Rhesus

Dog

Bird

Frog

0 10 20 30 40 50 60 70 Number of Amino Acid Differences in a Hemoglobin Polypeptide

Figure 1.10 Molecules reflect evolutionary patterns. 

Vertebrates that are more distantly related to humans have a greater number of amino acid differences in the hemoglobin polypeptide.

?

Inquiry question Where do you imagine a snake might fall on the graph? Why?

1.4 Core

Concepts in Biology

Learning Outcome 1. Discuss the core concepts that underlie the study of biology.

At the fundamental level of neurochemistry, the brain of a novice is largely the same as that of an expert. There are however, significant differences in how experts organize the information they collect over time. As you are starting to gather information about biology, it is worth considering how you can begin to organize this information in your mind like an expert thinker. You may be trying to organize the flood of information about biology by topics. The problem with this approach is that there are just too many topics for this to be successful. A better way to organize ideas in your mind is using a conceptual framework. Most disciplines, including biology, are based on information that is readily organized around concepts. You can think of concepts as a place in your mind to hold specific ideas that relate to many topics. For example, consider a hammer, a sunflower, and DNA. These seem quite disparate, but can actually be organized conceptually. A hammer has a long handle to create leverage and a heavy head to drive nails. Sunflowers have broad leaves that maximize their ability to absorb light for photosynthesis, and DNA has a 12 part I The Molecular Basis of Life

structure that allows storage of information. These descriptions can be organized into the concept “structure determines function”: the function of something arises from its form. When you encounter new information, you can fit it into a framework of core concepts like “structure determines function.” There has been a recent movement to emphasize core concepts in biology education. The authors applaud this and have incorporated this approach in this text. We have emphasized five core concepts: life is subject to chemical and physical laws; structure determines function; living systems transform energy and matter; living systems depend on information transactions; and evolution explains the unity and diversity of life. Core concepts are, by their very nature, high level and thus general. These are used to organize more specific secondary concepts, which in turn arise from observations, experiments, or descriptions of biological phenomena. For example, the core concept “structure determines function” could lead to the secondary concept “Genetic information is encoded in the structure of DNA.” This can then be used to organize a series of observations about the nature of genetic information and how it is used, such as these: “base pairing involves specific patterns of hydrogen bonds,” and “the genetic code consists of four nucleotides that are abbreviated: A,T,G,C,” and “DNA is used as a template to synthesize RNA,” and so on.

The five core concepts Life is subject to chemical and physical laws It may seem obvious, but it is important to emphasize that living systems operate according to known chemical and physical principles. For this reason, almost all introductory textbooks, including this one, begin with several sections on chemistry. This is because biological systems are the ultimate application of some very complex chemistry. However, there are no new chemical or physical laws to be found in biology, just the consistent application of familiar chemical principles and laws. This means that some knowledge of atomic structure, chemical bonding, thermodynamics, kinetics, and many other topics from basic chemistry and physics is crucial for understanding biological systems. It may seem that some physics and chemistry would only be relevant in the “cell and molecular” sections of the book, but in fact, those principles are applied throughout the book. The movement of water in plants depends on the basic chemistry of water, the kidney is an osmotic machine, energy flow and nutrient cycling in ecosystems are driven by thermodynamic laws, and the cycling of many important elements involves biogeochemical cycles.

Structure determines function A major unifying theme of biology is the relationship between structure and function. Said simply, the proper functioning of molecules, of cells, and of tissues and organs depends on their structure. Although this observation may seem trivial, it has far-reaching implications. When we know the function of a particular structure, we can infer the function of similar structures found in different contexts, such as in different organisms.

For example, suppose we know the structure of a human cell’s surface receptor for insulin, the hormone that controls the uptake of glucose. We then find a similar molecule in the membrane of a cell from a very different species, such as a worm. We might conclude that this membrane molecule acts as a receptor for an insulin-like molecule produced by the worm. In this way, we might be able to hypothesize an evolutionary relationship between glucose uptake in worms and in humans. When structure is altered, function is disrupted, with potential physiological consequences.

Living systems transform energy and matter From single cells to the highest level of biological organization, the biosphere, living systems have a constant need for energy. If we trace this all the way back, the original energy source for the biosphere is the sun. Without this energy, living systems would not exhibit their characteristic highly organized state. While this sounds simple, it means that the basic nature of life is to constantly transform both energy and matter. We break down “food” molecules for energy, then use this energy to build up other complex molecules. The energy from the sun is trapped by photosynthetic organisms, which use this energy to reduce CO2 and produce organic compounds. The rest of us, who need a constant source of energy and carbon, can oxidize these organic compounds back to CO2, releasing energy to drive the processes of life. As all of these energy transactions are inefficient, a certain amount of energy is also randomized as heat. This constant input of energy allows living systems to function far from thermodynamic equilibrium. At equilibrium, you are a pool of amino acids, nucleotides, and other small molecules, and not the complex dynamic system reading this sentence. Nonequilibrium systems also can exhibit the property of self-organization not seen in equilibrium systems. Macromolecular complexes, such as the spindle necessary for chromosome separation, can selforganize (figure 1.11). A flock of birds, a school of fish, and the bacteria in a biofilm all also display self-organization, exhibiting properties not seen in the individual parts alone.

Figure 1.11 The spindle. In this dividing cell, microtubules have organized themselves into a spindle (stained red), pulling each chromosome (stained blue) to the central plane of the dividing cell. Waheeb K. Heneen/Swedish University of Agricultural Sciences

Living systems depend on information transactions The most obvious form of information in living systems is the genetic information carried in every cell in the form of deoxyribonucleic acid (DNA). Each DNA molecule is formed from two long chains of building blocks, called nucleotides, wound around each other (figure 1.12). Four different nucleotides are found in DNA, and the sequence in which they occur encodes the information to make and maintain a cell. The continuity of life from one generation to the next—­ heredity—depends on the faithful copying of a cell’s DNA into daughter cells. The entire set of DNA instructions that specifies a cell is called its genome. The sequence of the human genome, 3 billion nucleotides long, was decoded in rough-draft form in 2001. However, the importance of information goes beyond genomes and their inheritance. Cells are highly complex nanomachines that receive, process, and respond to information. The

Figure 1.12 DNA, the genetic material. All organisms store their hereditary information as sequences of DNA subunits, much as this textbook stores information as sequences of alphabet letters.

LAGUNA DESIGN/Science Photo Library/Alamy Stock Photo

information stored in DNA is used to direct the synthesis of cellular components, and the particular set of components can differ from cell to cell. The way proteins fold in space is a form of information that is three-dimensional, and interesting properties emerge from the interaction of these shapes in macromolecular complexes. The control of gene expression allows the

1

chapter  The Science of Biology 13

differentiation of cell types in time and space, leading to changes over developmental time into different tissue types—even though all cells in an organism carry the same genetic information. Living systems are able to collect information about the environment, both internal and external, and then respond to this information. As you are reading this page, you are familiar with this process, but it also occurs at the level of cells, in terms of both single-celled organisms and the cells of multicellular organisms. Cells acquire information about their environment, send and receive signals, and respond to all of this information with signal transduction systems that can change cell morphology, behavior, or physiology (the subject of chapter 9).

Evolution explains the unity and diversity of life Biologists agree that all organisms alive today on Earth descended from a simple cellular organism that arose about 3.5 bya. Some of the characteristics of that organism have been preserved through evolutionary history into the present. The storage of hereditary information in DNA, for example, is common to all living things. The retention of these conserved characteristics in a long line of descent implies that they have a fundamental role in the success of the organism. A good example is provided by the homeodomain proteins, which are critical regulators of the process of development. Conserved characteristics can be seen in approximately 1850 homeodomain proteins, distributed among three kingdoms of organisms (figure 1.13). The homeodomain proteins are powerful developmental tools that evolved early; they have been used and modified to provide new forms. The unity of life that we see in certain key characteristics shared by many related life-forms contrasts with the incredible diversity of living things in the varied environments of Earth. The underlying unity of biochemistry and genetics argues that all life evolved from the same origin event. The incredible diversity of life we see today arose by evolutionary change, much of it visible in the fossil record.

Mus musculus (animal)

Saccharomyces cerevisiae (fungus)

MEIS

MATa2

PHO2 HB8

KN

HAT BEL1 Arabidopsis thaliana (plant)

GL2 MATa1

Saccharomyces cerevisiae (fungus)

PEM

PAX6

Arabidopsis thaliana (plant)

Mus musculus (animal)

Figure 1.13 Tree of homeodomain proteins. 

Homeodomain proteins are found in fungi (brown), plants (green), and animals (blue). Based on their sequence similarities, these 11 different homeodomain proteins (uppercase letters at the ends of branches) fall into two groups, with representatives from each kingdom in each group. That means, for example, the mouse homeodomain protein PAX6 is more closely related to fungal and flowering plant proteins, such as PHO2 and GL2, than it is to the mouse protein MEIS.

Learning Outcome Review 1.4

Understanding biology requires higher-level concepts. We are using five core concepts throughout the book: life is subject to chemical and physical laws, structure determines function, living systems transform energy and matter, living systems depend on information transactions, and evolution explains the unity and diversity of life. ■■

How do viruses fit into our definitions of living systems?

Chapter Review Soames Summerhays/Natural Visions

1.1 The Science of Life

Biology unifies much of natural science. The study of biological systems is interdisciplinary because solutions require many different approaches to solve a problem. Life defies simple definition. Although life is difficult to define, living systems have seven characteristics in common. They are composed of one or more cells; are complex and 14 part I The Molecular Basis of Life

highly ordered; can respond to stimuli; can grow, reproduce, and transmit genetic information to their offspring; need energy to accomplish work; can maintain relatively constant internal conditions (homeostasis); and are capable of evolutionary adaptation to the environment. Living systems show hierarchical organization. The hierarchical organization of living systems progresses from atoms to the biosphere. At each higher level, emergent properties arise that are greater than the sum of the parts.

1.2 The Nature of Science At its core, science is concerned with understanding the nature of the world by using observation and reasoning. Much of science is descriptive. Science is concerned with developing an increasingly accurate description of nature through observation and experimentation. Science uses both deductive and inductive reasoning. Deductive reasoning applies general principles to predict specific results. Inductive reasoning uses specific observations to construct general scientific principles. Hypothesis-driven science makes and tests predictions. Hypotheses are based on observations, and generate testable predictions. Experiments involve a test where a variable is manipulated, and a control where the variable is not manipulated. If the predictions cannot be verified the hypothesis is rejected. Reductionism breaks larger systems into their component parts. Reductionism attempts to understand a complex system by breaking it down into its component parts. It is limited because parts may act differently when isolated from the larger system.

Darwin proposed natural selection as a mechanism for evolution. Darwin noted that species produce many more offspring than will survive and reproduce. He observed that traits can be changed by artificial selection. Darwin proposed that individuals possessing traits that increase survival and reproductive success become more numerous in populations over time. Darwin called this descent with modification (natural selection). Alfred Russel Wallace independently came to the same conclusions. The predictions of natural selection have been tested. Natural selection has been tested using data from many fields. Among these are the fossil record; the age of the Earth, determined by rates of radioactive decay to be 4.5 billion years; genetic experiments showing that traits can be inherited as discrete units; comparative anatomy and the study of homologous structures; and molecular data that provide evidence for changes in DNA and proteins over time. Taken together, these findings strongly support evolution by natural selection. No data to conclusively disprove evolution have been found.

1.4 Core Concepts in Biology We use core concepts to organize information about the world around us. We introduce five core concepts to be used throughout this book, to help organize your thinking.

Biologists construct models to explain living systems. A model provides a way of organizing our thinking about a problem; models may also suggest experimental approaches.

Life is subject to chemical and physical laws. All living systems function based on the laws of chemistry and physics.

The nature of scientific theories. Scientists use the word theory in two main ways: as a proposed explanation for some natural phenomenon and as a body of concepts that explains facts in an area of study.

Structure determines function. The function of macromolecules is dictated by and dependent on their structure. Similarity of structure and function may indicate an evolutionary relationship.

Research can be basic or applied. Basic research extends the boundaries of what we know; applied research seeks to use scientific findings in practical areas such as agriculture, medicine, and industry.

1.3 An Example of Scientific Inquiry: Darwin

and Evolution

Darwin’s theory of evolution shows how a scientist develops a hypothesis and sets forth evidence, as well as how a scientific theory grows and gains acceptance. The idea of evolution existed prior to Darwin. A number of naturalists and philosophers had suggested living things had changed during Earth’s history. Darwin’s contribution was the concept of natural selection as a mechanism for evolutionary change. Darwin observed differences in related organisms. During the voyage of the H.M.S. Beagle, Darwin had an opportunity to observe worldwide patterns of diversity.

Living systems transform energy and matter. Living systems have a constant need for energy, which is ultimately provided by the sun. The nature of life is to constantly transform energy. We break down food molecules to provide energy to build up complex structures. Living systems depend on information transactions. Hereditary information found in the DNA molecule is passed on from one generation to the next. This information is read out to produce proteins, which themselves have information in their structures. Living systems can also acquire information about their environment. Evolution explains the unity and diversity of life. The underlying similarities in biochemistry and genetics support the contention that all life evolved from a single source. The diversity found in living systems arises by evolutionary change.

1

chapter  The Science of Biology 15

Visual Summary Methods of science

Biology

Living systems

a fo rm unified by

Observation

Hierarchy from

Reasoning include

ed fin de by

use

Shared characteristics

Cellular

Deductive

Cellular organization

Inductive Core concepts

include

Organism

Ordered complexity Sensitivity

Hypothesis testing

Reductionism

Modeling

include

Population

Growth, development, reproduction Energy use

leads to

Chemical and physical laws

Structure determines function

Energy transformations

Information transactions

Evolution

Homeostasis Evolutionary adaptation

Theory

Review Questions Soames Summerhays/Natural Visions

U N D E R S TA N D 1. Which of the following is NOT a property of life? a. b. c. d.

Energy utilization Movement Order Homeostasis

2. The process of inductive reasoning involves a. b. c. d.

the use of general principles to predict a specific result. the generation of specific predictions based on a belief system. the use of specific observations to develop general principles. the use of general principles to support a hypothesis.

3. A hypothesis in biology is best described as a. b. c. d.

a possible explanation of an observation. an observation that supports a theory. a general principle that explains some aspect of life. an unchanging statement that correctly predicts some aspect of life.

16 part I The Molecular Basis of Life

4. A scientific theory is a. b. c. d.

a guess about how things work in the world. a statement of how the world works that is supported by experimental data. a belief held by many scientists. Both a and c are correct.

5. The cell theory states that a. b. c. d.

cells are small. cells are highly organized. there is only one basic type of cell. all living things are made up of cells.

6. The molecule DNA is important to biological systems because a. b. c. d.

it can be replicated. it encodes the information for making a new individual. it forms a complex, double-helical structure. nucleotides form genes.

7. The organization of living systems is a. b. c. d.

linear with cells at one end and the biosphere at the other. circular with cells in the center. hierarchical with cells at the base, and the biosphere at the top. chaotic and beyond description.

8. The idea of evolution a. b. c. d.

was original to Darwin. was original to Wallace. predated Darwin and Wallace. Both a and b are correct.

A P P LY 1. What is the significance of Pasteur’s experiment to test the germ hypothesis? a. b. c. d.

It proved that heat can sterilize a broth. It demonstrated that cells can arise spontaneously. It demonstrated that some cells are germs. It demonstrated that cells can arise only from other cells.

2. Which of the following is NOT an example of reductionism? a. b. c. d.

Analysis of an isolated enzyme’s function in an experimental assay Investigation of the effect of a hormone on cell growth in a Petri dish Observation of the change in gene expression in response to specific stimulus An evaluation of the overall behavior of a cell

3. How is the process of natural selection different from that of artificial selection? a. b. c. d.

Natural selection produces more variation. Natural selection makes an individual better adapted. Artificial selection is a result of human intervention. Artificial selection results in better adaptations.

4. If you found a fossil for a modern organism next to the fossil of a dinosaur, this would a. b. c. d.

argue against evolution by natural selection. have no bearing on evolution by natural selection. indicate that dinosaurs may still exist. Both b and c are correct.

5. The theory of evolution by natural selection is a good example of how science proceeds because a. b. c. d.

it rationalizes a large body of observations. it makes predictions that have been tested by a variety of approaches. it represents Darwin’s belief of how life has changed over time. Both a and b are correct.

6. In which domain of life would you find only single-celled organisms? a. b. c. d.

Eukarya Bacteria Archaea Both b and c are correct.

7. Evolutionary conservation occurs when a characteristic is a. b. c. d.

important to the life of the organism. not influenced by evolution. no longer functionally important. found in more primitive organisms.

SYNTHESIZE 1. Exobiology is the study of life on other planets. In recent years, scientists have sent various spacecraft out into the galaxy in search of extraterrestrial life. Assuming that all life shares common properties, what should exobiologists be looking for as they explore other worlds? 2. The classic experiment by Pasteur (figure 1.4) tested the hypothesis that cells arise from other cells. In this experiment cell growth was measured following sterilization of broth in a swan-necked flask or in a flask with a broken neck. a. b. c. d.

Which variables were kept the same in these two experiments? How does the shape of the flask affect the experiment? Predict the outcome of each experiment based on the two hypotheses. Some bacteria (germs) are capable of producing heat-resistant spores that protect the cell and allow it to continue to grow after the environment cools. How would the outcome of this experiment have been affected if spore-forming bacteria were present in the broth?

1

chapter  The Science of Biology 17

2

CHAPTER

The Nature of Molecules and the Properties of Water Chapter Contents 2.1

The Nature of Atoms

2.2

Elements Found in Living Systems

2.3

The Nature of Chemical Bonds

2.4

Water: A Vital Compound

2.5

Properties of Water

2.6

Acids and Bases

Virtualphoto/Getty Images

Introduction

Visual Outline Chemistry of life

Atoms

Elements

make ke up p

defined by

Molecules

Atomic number

Water

is

Dissociates

Polar can form

opposite charges

Bonds

shared electrons Ionic Cl− Na+ Cl− Na+ Cl− Cl−

H

Hydrogen bonds

δ+ δ−

affect properties

Covalent unequal

equal

Na+

Na+ Cl−

H

Polar

Nonpolar

Cohesion

Adhesion

High specific head

High heat of vaporization

Less dense solid

Solvent

About 12.5 billion years ago (bya), an enormous explosion probably signaled the beginning of the universe. This explosion started a process of star building and planetary formation that eventually led to the formation of Earth, about 4.5 bya. Around 3.5 bya, life began on Earth and started to diversify. To understand the nature of life on Earth, we first need to understand the nature of the matter that forms the building blocks of all life. The earliest speculations about the world around us included this most basic question, “What is it made of?” The ancient Greeks recognized that larger things may be built of smaller parts. This concept was formed into a solid ­experimental scientific idea in the early 20th century, when physicists began trying to break atoms apart. From those humble b ­ eginnings to the huge particle accelerators used by the modern physicists of today, the picture of the atomic world emerges as ­fundamentally different from the tangible, macroscopic world around us.

To understand how living systems are assembled, we must first understand a little about atomic structure, about how atoms can be linked together by chemical bonds to make molecules, and about the ways in which these small molecules are joined together to make larger molecules, until finally we arrive at the structures of cells and then of organisms. Our study of life on Earth therefore begins with physics and chemistry. For many of you, this chapter will be a review of material encountered in other courses.

2.1

The Nature of Atoms

SCIENTIFIC THINKING Hypothesis: Atoms are composed of diffuse positive charge with embedded negative charge (electrons). Prediction: If alpha (α) particles, which are helium nuclei, are shot at a thin foil of gold, the α particles will not be deflected much by the diffuse positive charge or by the light electrons. Test: α particles are shot at a thin sheet of gold foil surrounded by a detector screen, which shows flashes of light when hit by the particles. 2. Most α particles pass through foil with little or no deflection.

1. α Particles are fired at gold foil target. Gold foil

Learning Outcomes 1. Define an element based on its composition. 2. Describe the relationship between atomic structure and chemical properties. 3. Explain where electrons are found in an atom.

Any substance in the universe that has mass and occupies space is defined as matter. All matter is composed of extremely small par­ ticles called atoms. Because of their size, atoms are difficult to study. In the early 20th century scientists carried out the first ex­ periments that revealed the physical nature of atoms (figure 2.1).

Atomic structure includes a central nucleus and orbiting electrons Objects as small as atoms can be visualized only indirectly, by using complex technology such as tunneling microscopy (fig­ ure 2.2). We now know a great deal about the complexities of atomic structure, but the simple view put forth in 1913 by the Danish physicist Niels Bohr provides a good starting point for understanding atomic theory. Bohr proposed that every atom possesses an orbiting cloud of tiny subatomic particles called electrons whizzing around a core, like the planets of a miniature solar system. At the center of each atom is a small, very dense nucleus formed of two other kinds of subatomic particles: ­protons and neutrons (figure 2.3).

α Particle source

Detector screen

3. Some α particles are deflected by more than 90°. Result: Most particles are not deflected at all, but a small percentage of particles are deflected at angles of 90° or more. Conclusion: The hypothesis is not supported. The large deflections observed led to a view of the atom as composed of a very small central region containing positive charge (the nucleus) surrounded by electrons. Further Experiments: How does the Bohr atom with its quantized energy for electrons extend this model?

Figure 2.1 Rutherford scattering experiment. Large-angle

scattering of α particles led Rutherford to propose the existence of the nucleus.

Atomic number Different atoms are defined by their atomic number, which is the number of protons. Atoms with the same atomic number have the same number of protons, exhibit the same chemical properties, and are said to be the same element. An element is defined as any sub­ stance that cannot be broken down to any other substance by ordi­ nary chemical means. Within the nucleus, the cluster of protons and neutrons is held together by a force that works only over short, subatomic distances. Each proton carries a positive (+) charge, and each neutron has no charge. Each electron carries a negative (−) charge. Typically, an atom has one electron for each proton and is thus electrically neu­ tral. The chemical behavior of an atom is due to the number and configuration of electrons, as we will see later in this section.

Figure 2.2 Scanning-tunneling microscope image. 

The scanning-tunneling microscope is a nonoptical imaging technique that allows atoms to be directly visualized. This image shows a lattice of silicon atoms in a silicon chip. Andrew Dunn/Alamy Stock Photo chapter

2 The Nature of Molecules and the Properties of Water 19

Hydrogen

Oxygen

1 Proton 1 Electron

8 Protons 8 Neutrons 8 Electrons

Earth’s gravitational force is greater than the Moon’s. The atomic mass of an atom is equal to the sum of the masses of its protons and neutrons. Atoms that occur naturally on Earth contain from 1 to 94 protons and up to 146 neutrons. The mass of atoms and subatomic particles is measured in units called daltons. To give you an idea of just how small these units are, note that it takes 602 million million billion (6.02 × 1023) daltons to make 1 gram (g). A proton weighs approximately 1 dal­ ton (actually 1.007 daltons), as does a neutron (1.009 daltons). In contrast, electrons weigh only 1/1840 of a dalton, so they contribute almost nothing to the overall mass of an atom.

Electrons a.

b. proton (positive charge)

electron (negative charge)

neutron (no charge)

Figure 2.3 Basic structure of atoms. All atoms have a

nucleus consisting of protons and neutrons, except hydrogen, the smallest atom, which usually has only one proton and no neutrons in its nucleus. Oxygen typically has eight protons and eight neutrons in its nucleus. In the simple “Bohr model” of atoms pictured here, electrons spin around the nucleus at a relatively far distance. a. Atoms are depicted as a nucleus with a cloud of electrons (not shown to scale). b. The electrons are shown in discrete energy levels. These are described in greater detail in the text.

Atomic mass The terms mass and weight are often used interchangeably, but they have slightly different meanings. Mass refers to the amount of a substance, but weight refers to the force gravity exerts on a sub­ stance. An object has the same mass whether it is on the Earth or the Moon, but its weight will be greater on the Earth because the

Figure 2.4 The three most abundant isotopes of carbon. Isotopes of a

particular element have different numbers of neutrons.

20 part I The Molecular Basis of Life

Carbon-12 6 Protons 6 Neutrons 6 Electrons

The positive charges in the nucleus of an atom are neutralized, or counterbalanced, by negatively charged electrons, which are located in regions called orbitals that lie at varying distances around the nucleus. Atoms with the same number of protons and electrons are electrically neutral—that is, they have no net charge, and are therefore called neutral atoms. Electrons are maintained in their orbitals by their attraction to the positively charged nucleus. Sometimes other forces overcome this attraction, and an atom loses one or more electrons. In other cases, atoms gain additional electrons. Atoms in which the number of electrons does not equal the number of protons are known as ions, and they are charged particles. An atom having more protons than electrons has a net positive charge and is called a cation. For example, an atom of sodium (Na) that has lost one electron becomes a sodium ion (Na+), with a charge of +1. An atom having fewer protons than electrons carries a net negative charge and is called an anion. A chlorine atom (Cl) that has gained one electron becomes a chloride ion (Cl−), with a charge of −1.

Isotopes Although all atoms of an element have the same number of pro­ tons, they may not all have the same number of neutrons. Atoms of a single element that possess different numbers of neutrons are called isotopes of that element. Most elements in nature exist as mixtures of different isotopes. Carbon (C), for example, has three isotopes, all contain­ ing six protons (figure 2.4). Over 99% of the carbon found in nature exists as an isotope that also contains six neutrons. Because the total mass of this isotope is 12 daltons (6 from protons plus 6 from neutrons), it is referred to as carbon-12 and is symbolized 12C. Most of the rest of the naturally occurring carbon is carbon-13, an Carbon-13 6 Protons 7 Neutrons 6 Electrons

Carbon-14 6 Protons 8 Neutrons 6 Electrons

isotope with seven neutrons. The rarest carbon isotope is carbon-14, with eight neutrons. Unlike the other two isotopes, carbon-14 is unstable: this means that its nucleus tends to break up into ele­ ments with lower atomic numbers. This nuclear breakup, which emits a significant amount of energy, is called radioactive decay, and isotopes that decay in this fashion are radioactive isotopes. Some radioactive isotopes are more unstable than others, and therefore they decay more readily. For any given isotope, however, the rate of decay is constant. The decay time is usually expressed as the half-life, the time it takes for one-half of the atoms in a sample to decay. Carbon-14, for example, often used in the carbon dating of fossils and other materials, has a half-life of 5730 years. A sample of carbon containing 1 g of c­ arbon-14 today would contain 0.5 g of carbon-14 after 5730 years, 0.25 g 11,460 years from now, 0.125 g 17,190 years from now, and so on. By determining the ratios of the different isotopes of carbon and other elements in biological samples and in rocks, scientists are able to accurately determine when these materials formed. Radioactivity has many useful applications in modern biol­ ogy. Radioactive isotopes are one way to label, or “tag,” a specific molecule and then follow its progress, either in a chemical reaction

Electron Shell Diagram

Corresponding Electron Orbital

Energy level K

One spherical orbital (1s)

a. Electron Shell Diagram

or in living cells and tissue. The downside, however, is that the energetic subatomic particles emitted by radioactive substances have the potential to severely damage living cells, producing genetic mutations and, at high doses, cell death. Consequently, ex­ posure to radiation is carefully controlled and regulated. Scientists who work with radio­activity follow strict handling protocols and wear radiation-­sensitive badges to monitor their exposure over time to help ensure a safe level of exposure.

Electrons determine the chemical behavior of atoms The key to the chemical behavior of an atom lies in the number and arrangement of its electrons in their orbitals. The Bohr model of the atom shows individual electrons as following distinct circular orbits around a central nucleus. The trouble with this simple pic­ ture is that it doesn’t reflect reality. Modern physics indicates that we cannot pinpoint the position of any individual electron at any given time. In fact, an electron could be anywhere, from close to the nucleus to infinitely far away from it. A particular electron, however, is more likely to be in some areas than in others. An orbital is defined as the area around a nu­ cleus where an electron is most likely to be found. These orbitals represent probability distributions for electrons—that is, regions more likely to contain an electron. Some electron orbitals near the nucleus are spherical (s orbitals), whereas others are dumbbellshaped (p orbitals) (figure 2.5). Still other orbitals, farther away from the nucleus, may have different shapes. Regardless of its shape, no orbital can contain more than two electrons. Almost all of the volume of an atom is empty space. This is because the electrons are usually far away from the nucleus, rela­ tive to its size. If the nucleus of an atom were the size of a golf ball, the orbit of the nearest electron would be a mile away. Consequently,

Corresponding Electron Orbitals y

z

Energy level L x

One spherical orbital (2s)

Three dumbbell-shaped orbitals (2p)

b. Electron Shell Diagram

Electron Orbitals

Figure 2.5 Electron orbitals. a. The lowest energy level, or

y z

x Neon

c.

electron shell—the one nearest the nucleus—is level K. It is occupied by a single s orbital, referred to as 1s. b. The next highest energy level, L, is occupied by four orbitals: one s orbital (referred to as the 2s orbital) and three p orbitals (each referred to as a 2p orbital). Each orbital holds two paired electrons with opposite spin. Thus, the K level is populated by two electrons, and the L level is populated by a total of eight electrons. c. The neon atom shown has the L and K energy levels completely filled with electrons and is thus unreactive. chapter

2 The Nature of Molecules and the Properties of Water 21

the nuclei of two atoms never come close enough in nature to inter­ act with each other. It is for this reason that an atom’s electrons, not its protons or neutrons, determine its chemical behavior, and it also explains why the isotopes of an element, all of which have the same arrangement of electrons, behave the same way chemically.

Atoms contain discrete energy levels Because electrons are attracted to the positively charged nucleus, it takes work to keep them in their orbitals, just as it takes work to hold a grapefruit in your hand against the pull of gravity. The for­ mal definition of energy is the ability to do work. The grapefruit held above the ground is said to possess potential energy because of its position. If you release it, the grape­ fruit falls, and its potential energy is reduced. On the other hand, if you carried the grapefruit to the top of a building, you would in­ crease its potential energy. Electrons also have a potential energy that is related to their position. To oppose the attraction of the nu­ cleus and move the electron to a more distant orbital requires an input of energy, which results in an electron with greater potential energy. The chlorophyll that makes plants green captures energy from light during photosynthesis in this way. As you’ll see in chap­ ter 8, light energy excites electrons in the chlorophyll molecule. Moving an electron closer to the nucleus has the opposite effect: energy is released, usually as radiant energy (heat or light), and the electron ends up with less potential energy (figure 2.6). One of the initially surprising aspects of atomic structure is that electrons within the atom have discrete energy levels. These discrete levels correspond to quanta (singular, quantum, which means “specific amount of energy”). To use the grapefruit analogy again, it is as though a grapefruit could be raised only to particular floors of a building. Every atom exhibits a ladder of potential en­ ergy values, a discrete set of orbitals at particular energetic “dis­ tances” from the nucleus. Because the amount of energy an electron possesses is re­ lated to its distance from the nucleus, electrons that are the same distance from the nucleus have the same energy, even if they occupy different orbitals. Such electrons are said to occupy the same energy level. The energy levels are denoted with letters K, L, M, and so on (figure 2.6). Be careful not to confuse energy levels, which are drawn as rings to indicate an electron’s energy, with orbitals, which have a variety of three-dimensional shapes Energy released

and indicate an electron’s most likely l­ ocation. Electron orbitals are arranged so that as they are filled, this fills each energy level in successive order. This filling of orbitals and energy levels is what is responsible for the chemical reactivity of elements. During some chemical reactions, electrons are transferred from one atom to another. In such reactions, the loss of an electron is called oxidation, and the gain of an electron is called reduction.

+

+

Oxidation

Reduction

Notice that when an electron is transferred in this way, it keeps its energy of position. In organisms, chemical energy is stored in high-energy electrons that are transferred from one atom to another in reactions involving oxidation and reduction (described in chapter 7). When the processes of oxidation and reduction are coupled, which often happens, one atom or molecule is oxidized, while another is reduced in the same reaction. We call these combinations redox reactions.

Learning Outcomes Review 2.1

An atom consists of a nucleus of protons and neutrons surrounded by a cloud of electrons. The number of protons in an atom is the atomic number, and atoms with the same atomic number constitute an element. Atoms of an element that have different numbers of neutrons are called isotopes. Electrons, which determine the chemical behavior of an element, are located in orbitals, and are also found only at discrete energy levels. No orbital can contain more than two electrons, but each energy level consists of multiple orbitals, and thus contains many electrons with the same energy. ■■ ■■

If the number of protons exceeds the number of neutrons, is the charge on the atom positive or negative? If the number of protons exceeds electrons?

Energy absorbed

Nucleus K L M N

N M L K Nucleus

Figure 2.6 Atomic energy levels. Electrons have energy of position. When an atom absorbs energy, an electron moves to a higher energy level, farther from the nucleus. When an electron falls to lower energy levels, closer to the nucleus, energy is released. The first two energy levels are the same as shown in figure 2.5. 22 part I The Molecular Basis of Life

an element reflects how many of the eight positions are filled. Elements with eight electrons in their outer energy level (two for helium) are inert, or nonreactive. These elements, helium (He), neon (Ne), argon (Ar), and so on, are called the noble gases. In contrast, elements with only seven electrons in their outer energy level, such as fluorine (F), chlorine (Cl), and bromine (Br), are highly reactive. They tend to gain the extra electron needed to fill the energy level. Elements with only one electron in their outer energy level, such as lithium (Li), sodium (Na), and potassium (K), are also very reactive, but they tend to lose the single electron in their outer level. These observations led to the so-called octet rule, or rule of eight (Latin octo, “eight”): atoms tend to establish completely full outer energy levels. For main group elements of the periodic table, the rule of eight requires one filled s orbital and three filled p orbit­ als (figure 2.8). The exception is He, which needs only two elec­ trons to fill the 1s orbital. Most chemical behavior of biological interest can be predicted with this simple rule, combined with the tendency of atoms to balance positive and negative charges. For instance, in section 2.3 we will see that sodium ions, Na+, and chloride ions, Cl−, can react to form table salt, NaCl. Of the 94 naturally occurring elements, only 12 (O, C, H, N, P, S, Na, K, Ca, Mg, Fe, Cl) are found in living systems in more than trace amounts (0.01% or higher). These elements all have atomic numbers less than 21, and thus low atomic masses. The distribution of elements in living systems is obviously not random. The most common elements in cells are not the elements that are the most abundant in the Earth’s crust. Of these 12 common elements, the first 4 elements (oxygen, carbon, hydrogen, and nitrogen) make up 96.3% of the weight of your body. The majority of molecules found in your body are com­ pounds of carbon, which we call organic compounds. These

2.2  Elements

Found in Living Systems

Learning Outcomes 1. Relate atomic structure to the periodic table of the elements. 2. List the important elements found in living systems.

Ninety-four elements occur naturally, each with a different number of protons and a different arrangement of electrons. When the 19th-century Russian chemist Dmitri Mendeleev arranged the known elements in a table according to their atomic number, he discovered one of the great generalizations of science: the elements exhibit a pattern of chemical properties that repeats itself in groups of eight. This periodically repeating pattern lent the table its name: the periodic table of elements (figure 2.7).

The periodic table displays elements according to atomic number and properties The eight-element periodicity that Mendeleev found is based on the interactions of the electrons in the outermost energy level of the different elements. We call these v­ alence electrons, and their inter­ actions are the basis for the differing chemical properties of ele­ ments. For most of the atoms important to life, this valence shell can contain no more than eight electrons; the chemical behavior of 1

3

Li

11

1

20

37

55

atomic number chemical symbol

5

B

13

12

Mg Ca 38

Sr

Sc 39

Y

Ti 40

Zr

V

41

Nb 73

24

Cr 42

Mo

Mn 43

Tc

44

Co 45

Ru

Rh

28

Ni 46

Pd

Cu 47

Ag

Zn 48

Cd

Ga 49

In

32

Ge 50

Sn

As 51

Sb

S

Cl

Ar

34

35

36

Se

Br

52

53

Te

Pt

Au

Hg

Tl

Pb

Bi

Po

At

Rn

87

88

89

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

Rf

Db

Sg

Bh

Hs

Mt

Ds

Rg

Cn

Nh

Fl

Mc

Lv

Ts

58

59

60

61

62

63

64

65

66

67

68

69

70

(Actinide series)

Th

90

91

92

Pa

U

93

Np

94

Pu

95

96

Am Cm

97

Bk

Dy 98

Cf

Ho 99

Es

84

Er 100

Tm

Fm

101

Md

a.

85

54

Xe

Ir

Tb

83

I

Kr

77

Gd

82

33

18

76

Eu

81

P

17

Os

Pm Sm

80

31

16

Re

Nd

79

30

15

10

Ne

W

Pr

78

29

N

Ta

Ce

75

Fe

27

F

Hf

(Lanthanide series)

74

25

Si

9

7

La Ac

72

23

C

Ba Ra

57

22

26

Al

6 14

He

Cs Fr

56

21

2

O

Be

19

Rb

H

4

Na K

8

Key

H

86

Oxygen (O)

C

Carbon (C)

H

Hydrogen (H)

N

Nitrogen (N)

Na Sodium (Na) Cl

Chlorine (Cl)

Ca Calcium (Ca) P

Phosphorus (P)

Og

K

Potassium (K)

71

S

Sulfur (S)

Fe

Iron (Fe)

Yb

Lu

102

103

No

O

Lr

Mg Magnesium (Mg)

b.

Figure 2.7 Periodic table of the elements. a. In this representation, the frequency of elements that occur in the Earth’s crust is indicated by the height of the block. Elements shaded in green are found in living systems in more than trace amounts. b. The 12 common elements found in living systems are listed in descending order of abundance, shown in colors that will be used throughout the text. chapter

2 The Nature of Molecules and the Properties of Water 23

Nonreactive

Reactive

TA B L E 2 .1

2 protons 2 neutrons 2 electrons

7 protons 7 neutrons 7 electrons

Name

K K

L 2+

7+

Helium

Nitrogen

Figure 2.8 Electron energy levels for helium and nitrogen. Green balls represent electrons, blue ball represents

Bonds and Interactions Basis of Interaction

Strength

Covalent bond

Sharing of electron pairs

Strong

Ionic bond

Attraction of opposite charges

Hydrogen bond

Sharing of H atom

Hydrophobic interaction

Forcing of hydrophobic portions of molecules together in presence of polar substances

van der Waals attraction

Weak attractions between atoms due Weak to oppositely polarized electron clouds

the nucleus with number of protons indicated by number of (+) charges. Note that the helium atom has a filled K shell and is thus unreactive, whereas the nitrogen atom has five electrons in the L shell, three of which are unpaired, making it reactive.

by chemical bonds; these bonds can result when atoms with opposite charges attract each other (ionic bonds), when two atoms share one or more pairs of electrons (covalent bonds), or when atoms interact in other ways (table 2.1). We will start by examining ionic bonds, which form when atoms with opposite electrical charges (ions) attract.

organic compounds contain primarily these four elements (OCHN). Since water is the most common molecule in your body, oxygen, not carbon, is the most abundant element in your body. Some trace elements, such as zinc (Zn) and iodine (I), play crucial roles in liv­ ing processes even though they are present in tiny amounts. Iodine deficiency, for example, can lead to enlargement of the thyroid gland, causing a bulge at the neck called a goiter.

Ionic bonds form crystals Common table salt, the molecule sodium chloride (NaCl), is a lat­ tice of ions in which the atoms are held together by ionic bonds (figure 2.9). Sodium has 11 electrons: 2 in the inner e­ nergy level

Learning Outcomes Review 2.2

The periodic table shows the elements in terms of atomic number and repeating chemical properties. Only 12 elements are found in significant amounts in living organisms: O, C, H, N, P, S, Na, K, Ca, Mg, Fe, and Cl. ■■

Na+

Na

Sodium ion (+)

Sodium atom

Why are the noble gases more stable than other elements in the periodic table?

Cl −

Cl

2.3  The

Nature of Chemical Bonds

Chloride ion (−)

Chlorine atom

a.

Learning Outcomes 1. Predict which elements are likely to form ions. 2. Explain how molecules are formed from atoms joined by covalent bonds. 3. Contrast polar and nonpolar covalent bonds.

A group of atoms held together by energy in a stable association is called a molecule. When a molecule contains atoms of more than one element, it is called a compound. The atoms in a molecule are joined 24 part I The Molecular Basis of Life

Figure 2.9 The formation of ionic bonds by sodium chloride. 

Cl −

Na+

a. When a sodium atom donates an electron to a chlorine atom, the sodium atom is oxidized and the chlorine atom Na+ Cl − reduced. This produces a positively charged sodium ion, and a negatively Na+ Cl − charged chloride ion. b. The electrostatic attraction of oppositely charged ions leads to the formation of a lattice of Na+ and Cl−. b. NaCl crystal

Cl − Na+ Cl −

(K), 8 in the next level (L), and 1 in the outer (valence) level (M). The single, unpaired valence electron has a strong tendency to join with another unpaired electron in another atom. A stable configuration can be achieved if the valence electron is lost to another atom that also has an unpaired electron. The loss of this electron results in the formation of a positively charged sodium ion, Na+. The chlorine atom has 17 electrons: 2 in the K level, 8 in the L level, and 7 in the M level. As you can see in figure 2.9, one of the orbitals in the outer energy level has an unpaired electron (red circle). The addition of another electron fills that level and causes a negatively charged chloride ion, Cl−, to form. When placed together, metallic sodium and gaseous chlo­ rine react swiftly and explosively, as the sodium atoms are oxi­ dized, donating electrons to chlorine atoms, reducing them, and forming Na+ and Cl− ions. Because opposite charges attract, the Na+ and Cl− remain associated in an ionic compound, NaCl, which is electrically neutral. The electrical attractive force hold­ ing NaCl together, however, is not directed specifically between individual Na+ and Cl− ions, and no individual sodium chloride molecules form. Instead, the force exists between any one ion and all neighboring ions of the opposite charge. The ions aggre­ gate in a crystal matrix with a precise geometry. Such aggrega­ tions are what we know as salt crystals. If a salt such as NaCl is placed in water, the electrical attraction of the water molecules disrupts the forces holding the ions in their crystal matrix, caus­ ing the salt to dissolve into a roughly equal mixture of free Na+ and Cl− ions. Because living systems always include water, ions are more important than ionic crystals. Important ions in biological systems include Ca2+, which is involved in cell signaling, K+ and Na+, which are involved in the conduction of nerve impulses.

Covalent bonds build stable molecules Covalent bonds form when two atoms share one or more pairs of valence electrons. Consider gaseous hydrogen (H2) as an example. Each hydrogen atom has an unpaired electron and an unfilled outer energy level; for these reasons, the hydrogen atom is unstable. However, when two hydrogen atoms are in close association, each atom’s electron is attracted to both nuclei. In effect, the nuclei are able to share their electrons. The result is a diatomic (two-atom) molecule of hydrogen gas. The molecule formed by the two hydrogen atoms is stable for three reasons: 1. It has no net charge. The diatomic molecule formed as a result of this sharing of electrons is not charged because it still contains two protons and two electrons. 2. The octet rule is satisfied. Each of the two hydrogen atoms can be considered to have two orbiting electrons in its outer energy level. This state satisfies the octet rule, because each shared electron orbits both nuclei and is included in the outer energy level of both atoms. 3. It has no unpaired electrons. The bond between the two atoms also pairs the two free electrons. Unlike ionic bonds, covalent bonds are formed between two individual atoms, giving rise to true, discrete molecules.

The strength of covalent bonds The strength of a covalent bond depends on the number of shared electrons. Thus double bonds, which satisfy the octet rule by al­ lowing two atoms to share two pairs of electrons, are stronger than single bonds, in which only one electron pair is shared. In practical terms, more energy is required to break a double bond than to break a single bond. The strongest covalent bonds are triple bonds, such as those that link the two nitrogen atoms of nitrogen gas mol­ ecules (N2). Covalent bonds are represented in chemical formulas as lines connecting atomic symbols. Each line between two bonded atoms represents the sharing of one pair of electrons. The structural formulas of hydrogen gas and oxygen gas are H−H and O=O, respectively, and their molecular formulas are H2 and O2. The structural formula for N2 is N≡N. covalent bond

Single covalent bond hydrogen gas H

H

Double covalent bond oxygen gas O

O

N

H

O

O

N

N

O2

Triple covalent bond nitrogen gas N

H

H2

N2

Molecules with several covalent bonds A vast number of biological compounds are composed of more than two atoms. An atom that requires two, three, or four addi­ tional electrons to fill its outer energy level completely may ac­ quire them by sharing its electrons with two or more other atoms. For example, the carbon atom (C) contains six electrons, four of which are in its outer energy level and are unpaired. To satisfy the octet rule, a carbon atom must form four covalent bonds. Because four covalent bonds may form in many ways, carbon at­ oms are found in many different kinds of molecules. CO2 (carbon dioxide), CH4 (methane), and C2H5OH (ethanol) are just a few examples.

Polar and nonpolar covalent bonds Atoms differ in their affinity for electrons, a property called electronegativity. In general, electronegativity increases left to right across a row of the periodic table and decreases down the column. Thus the elements in the upper-right corner have the highest electronegativity. For bonds between identical atoms, for example, between two hydrogen or two oxygen atoms, the affinity for electrons is obviously the same, and the electrons are equally shared. Such chapter

2 The Nature of Molecules and the Properties of Water 25

bonds are termed nonpolar. The resulting compounds (H2 or O2) are also referred to as nonpolar. For atoms that differ greatly in electronegativity, electrons are not shared equally. The shared electrons are more likely to be closer to the atom with greater electronegativity, and less likely to be near the atom of lower electronegativity. In this case, although the molecule is still electrically neutral (same number of protons as electrons), the distribution of charge is not uniform. This unequal distribution results in regions of partial negative charge near the more electronegative atom, and regions of partial positive charge near the less electronegative atom. Such bonds are termed polar covalent bonds, and the molecules polar molecules. When draw­ ing polar molecules, these partial charges are usually symbolized by the lowercase Greek letter delta (δ). The partial charge seen in a polar covalent bond is relatively small—far less than the unit charge of an ion. For biological molecules, we can predict polarity of bonds by knowing the relative electronegativity of a small num­ ber of important atoms (table 2.2). Notice that although C and H differ slightly in electronegativity, this small difference is negligi­ ble, and C—H bonds are considered nonpolar. Because of its importance in the chemistry of water, we will explore the nature of polar and nonpolar molecules in section 2.4. Water (H2O) is a polar molecule with electrons more concentrated around the oxygen atom.

Chemical reactions alter bonds The formation and breaking of chemical bonds, which is the es­ sence of chemistry, is termed a chemical reaction. All chemical reactions involve the shifting of atoms from one molecule or ionic compound to another, without any change in the number or identity of the atoms. For convenience, we refer to the original molecules before the reaction starts as reactants, and the mole­ cules resulting from the chemical reaction as p ­roducts. For example: 6H2O + 6CO2 ⟶ C6H12O6 + 6O2

reactants ⟶ products

You may recognize this reaction as a simplified form of the photo­ synthesis reaction, in which water and carbon dioxide are com­ bined to produce glucose and oxygen. Most animal life ultimately depends on this reaction, which takes place in plants. (Photo­ synthetic reactions will be discussed in detail in chapter 8.)

TA B L E 2 . 2

Relative Electronegativities of Some Important Atoms

Atom

Electronegativity

O

3.5

N

3.0

C

2.5

H

2.1

26 part I The Molecular Basis of Life

The extent to which chemical reactions occur is influenced by three important factors: 1. Temperature. Heating the reactants increases the rate of a reaction because the reactants collide with one another more often. (Care must be taken that the temperature is not so high that it destroys the molecules.) 2. Concentration of reactants and products. Reactions proceed more quickly when more reactants are available, allowing more frequent collisions. An accumulation of products typically slows the reaction and, in reversible reactions, may speed the reaction in the reverse direction. 3. Catalysts. A catalyst is a substance that increases the rate of a reaction. It doesn’t alter the reaction’s equilibrium between reactants and products, but it does shorten the time needed to reach equilibrium, often dramatically. In living systems, proteins called enzymes catalyze almost every chemical reaction. Many reactions in nature are reversible. This means that the products may themselves be reactants, allowing the reaction to proceed in reverse. We can write the preceding reaction in the reverse order: C6H12O6 + 6O2 ⟶ 6H2O + 6CO2

reactants ⟶ products

This reaction is a simplified version of the oxidation of glucose by cellular respiration, in which glucose is broken down into water and carbon dioxide in the presence of oxygen. Virtually all organ­ isms carry out forms of glucose oxidation; details are covered later, in chapter 7.

Learning Outcomes Review 2.3

An ionic bond is an attraction between ions of opposite charge in an ionic compound. A covalent bond is formed when two atoms share one or more pairs of electrons. Complex biological compounds are formed in large part by atoms that can form one or more covalent bonds: O, C, H, and N. A polar covalent bond is formed by unequal sharing of electrons. Nonpolar bonds exhibit equal sharing of electrons. ■■

How is a polar covalent bond different from an ionic bond?

2.4 Water:

A Vital Compound

Learning Outcomes 1. Relate how the structure of water leads to hydrogen bonds. 2. Describe water’s cohesive and adhesive properties.

Of all the common molecules, only water exists as a liquid at the relatively low temperatures that prevail on the Earth’s s­urface. Three-fourths of the Earth is covered by liquid water (figure 2.10).

a. Solid

b. Liquid

c. Gas

Figure 2.10 Water takes many forms. a. When water cools below 0°C, it forms beautiful crystals, familiar to us as snow and ice. b. Ice turns to liquid when the temperature is above 0°C. c. Liquid water becomes steam when the temperature rises above 100°C, as seen in this hot spring at Yellowstone National Park.  (a) Glen Allison/Photodisc/Getty Images; (b) moodboard/Glow Images; (c) blickwinkel/S. Meyers/Alamy Stock Photo

When life was beginning, water provided a medium in which other molecules could move around and interact, without being held in place by strong covalent or ionic bonds. Life evolved in water for 2 billion years before spreading to land. And even today, life is in­ extricably tied to water. About two-thirds of any organism’s body is composed of water, and all organisms require a water-rich envi­ ronment, either inside or outside them, for growth and reproduc­ tion. It is no accident that tropical rainforests are bursting with life, whereas dry deserts appear almost lifeless except when water becomes temporarily plentiful, such as after a rainstorm.

Water’s structure facilitates hydrogen bonding Water has a simple molecular structure, consisting of an oxygen atom bound to two hydrogen atoms by two single covalent bonds (figure 2.11). The resulting molecule is stable, has no unpaired

Bohr Model

Ball-and-Stick Model

δ+ +

δ+

δ−

104.5

8p 8n +

δ+ δ−

δ− δ−

δ+

a.

Figure 2.11 Water has a simple molecular structure. 

b. Space-Filling Model

a. Each water molecule is δ+ composed of one oxygen atom δ− and two hydrogen atoms. The + δ oxygen atom shares one electron with each hydrogen atom. b. The c. greater electronegativity of the oxygen atom makes the water molecule polar: water carries two partial negative charges (δ−) near the oxygen atom and two partial positive charges (δ+), one on each hydrogen atom. c. Space-filling model shows what the molecule would look like if it were visible.

electrons, and carries no net electrical charge. The electronegativ­ ity of O is much greater than that of H (see table 2.2), so the bonds between these atoms are highly polar. The polarity of water mole­ cules underlies water’s chemistry and the chemistry of life. Consider the shape of a water molecule. Its two covalent bonds have a partial charge at each end: shown as δ− at the oxygen end and δ+ at the hydrogen end. The most stable arrangement of these charges is a tetrahedron (a pyramid with a triangle as its base), in which the two negative and two positive charges are ap­ proximately equidistant from one another with the oxygen atom at the center of the tetrahedron (figure 2.11b). This arrangement of partial charges allows water molecules to interact forming weak chemical associations called hydrogen bonds. The partially negative (δ−) O atom in one molecule is at­ tracted to the partially positive (δ+) H atoms of other water mole­ cules (figure 2.12). In all phases, every water molecule is interacting with multiple other water molecules, and as we shall see, hydrogen bonding leads to some interesting properties. Hydrogen bonding is not limited to water molecules. Any molecule with H attached to a more electronegative atom, usually oxygen (O) or nitrogen (N), can potentially form hydrogen bonds. While these are too weak to form stable molecules by themselves, like Velcro they can form close associations by the sum of many weak interactions. A hydrogen bond has only 5–10% the strength of a covalent bond, but hydrogen bonds are critical for DNA and protein structure, and thus underlie much of the organization of living systems.

Water molecules are cohesive The hydrogen bonding of water means that water molecules are attracted to one another—that is, water is cohesive. Each hydro­ gen bond is individually very weak and transient, lasting on average only a hundred-billionth (10−11) of a second. The cu­ mulative effects of large numbers of these bonds, however, can be enormous. Water forms an abundance of hydrogen bonds, which are responsible for many of its important physical prop­ erties (table 2.3). Water’s cohesion is responsible for its being a liquid, not a gas, at moderate temperatures. The cohesion of liquid water is also responsible for its surface tension. Small insects can walk on water (figure 2.13) because at the air–water interface, all the surface water molecules are hydrogen-bonded to molecules be­ low them. chapter

2 The Nature of Molecules and the Properties of Water 27

Hydrogen atom

Water molecule

δ+ Hydrogen bond

δ−

Oxygen atom

a.

Hydrogen atom Hydrogen bond δ+ δ− An organic molecule Oxygen atom

b.

Figure 2.12 Structure of a hydrogen bond. a. Hydrogen

bond between two water molecules. b. Hydrogen bond between an organic molecule (n-butanol) and water. H in n-butanol forms a hydrogen bond with oxygen in water. This kind of hydrogen bond is possible any time H is bound to a more electronegative atom (see table 2.2).

Water molecules are adhesive

Figure 2.13 Cohesion. Some insects, such as this water strider, literally walk on water. Because the surface tension of the water is greater than the force of one foot, the strider glides atop the surface of the water rather than sinking. The high surface tension of water is due to hydrogen bonding between water molecules. Herman Eisenbeiss/Science Source

it upward, is stronger than the force of gravity, pulling it down­ ward. The narrower the tube, the greater the electrostatic forces between the water and the glass, and the higher the water rises (figure 2.14). We will see how plants use this principle to transport water in chapter 36.

The polarity of water causes it to be attracted to other polar mole­ cules as well. This attraction for other polar substances is called adhesion. Water adheres to any substance with which it can form hydrogen bonds. This property explains why substances contain­ ing polar molecules get “wet” when they are immersed in water, but those that are composed of nonpolar molecules (such as oils) do not. The attraction of water to substances that have electrical charges on their surface is responsible for capillary action. If a glass tube with a narrow diameter is lowered into a beaker of wa­ ter, the water will rise in the tube above the level of the water in the beaker, because the adhesion of water to the glass surface, drawing

TA B L E 2 . 3

Figure 2.14 Adhesion. Capillary action causes the water within a narrow tube to rise above the surrounding water level; the adhesion of the water to the glass surface, which draws water upward, is stronger than the force of gravity, which tends to pull it down. The narrower the tube, the greater the surface area available for adhesion for a given volume of water, and the higher the water rises in the tube.

The Properties of Water

Property

Explanation

Example of Benefit to Life

Cohesion

Hydrogen bonds hold water molecules together.

Leaves pull water upward from the roots; seeds swell and germinate.

High specific heat

Hydrogen bonds absorb heat when they break and release heat when they form, minimizing temperature changes.

Water stabilizes the temperature of organisms and the environment.

High heat of vaporization

Many hydrogen bonds must be broken for water to evaporate.

Evaporation of water cools body surfaces.

Lower density of ice

Water molecules in an ice crystal are spaced relatively far apart because of hydrogen bonding.

Because ice is less dense than water, lakes do not freeze solid, allowing fish and other life in lakes to survive the winter.

Solubility

Polar water molecules are attracted to ions and polar compounds, making these compounds soluble.

Many kinds of molecules can move freely in cells, permitting a diverse array of chemical reactions.

28 part I The Molecular Basis of Life

Learning Outcomes Review 2.4

Because of its polar covalent bonds, water can form hydrogen bonds with itself and with other polar molecules. Hydrogen bonding is responsible for water’s cohesion, the force that holds water molecules together, and its adhesion, which is its ability to “stick” to other polar molecules. Capillary action results from both of these properties. ■■

If water were made of C and H instead of H and O, would it still be cohesive and adhesive? Why or why not?

2.5

Properties of Water

Learning Outcomes 1. Illustrate how hydrogen bonding affects the properties of water. 2. Explain the relevance of water’s unusual properties for living systems. 3. Identify the dissociation products of water.

Water moderates temperature through two properties: its high spe­ cific heat and its high heat of vaporization. Water also has the un­ usual property of being less dense in its solid form, ice, than as a liquid. Water acts as a solvent for polar molecules and exerts an organizing effect on nonpolar molecules. All these properties re­ sult from its polar nature.

Water’s high specific heat helps maintain temperature The temperature of any substance is a measure of how rapidly its individual molecules are moving. In the case of water, a large input of thermal energy is required to break the many hydrogen bonds that keep individual water molecules from moving about. There­ fore, water is said to have a high specific heat, which is defined as the amount of heat 1 g of a substance must absorb or lose to change its temperature by 1 degree Celsius (°C). Specific heat measures the extent to which a substance resists changing its temperature when it absorbs or loses heat. Because polar substances tend to form hydro­ gen bonds, the more polar it is, the higher is its specific heat. The specific heat of water (1 calorie/g/°C) is twice that of most carbon compounds and nine times that of iron. Only ammonia, which is more polar than water and forms very strong hydrogen bonds, has a higher specific heat than water (1.23 cal/g/°C). Still, only 20% of the hydrogen bonds are broken as water heats from 0° to 100°C. Because of its high specific heat, water heats up more slowly­ than almost any other compound and holds its temperature longer. Because organisms have a high water content, water’s high spe­ cific heat allows them to maintain a relatively constant internal temperature. The heat generated by the chemical reactions inside cells would destroy the cells if not for the absorption of this heat by the water within them.

Water’s high heat of vaporization facilitates cooling The heat of vaporization is defined as the amount of energy re­ quired to change 1 g of a substance from a liquid to a gas. A consid­ erable amount of heat energy (586 cal) is required to accomplish this change in water. As water changes from a liquid to a gas it requires energy (in the form of heat) to break its many hydrogen bonds. The evaporation of water from a surface cools that surface. Many organ­ isms dispose of excess body heat by evaporative cooling, for exam­ ple, through sweating in humans and many other vertebrates.

Solid water is less dense than liquid water At low temperatures, water molecules are locked into a ­crystal-­like lattice of hydrogen bonds, forming solid ice (see figure 2.10a). Interestingly, ice is less dense than liquid water because the hydro­ gen bonds in ice space the water molecules relatively far apart. This unusual feature enables icebergs to float. If water did not have this property, nearly all bodies of water would be ice, with only the shallow surface melting every year. The buoyancy of ice is impor­ tant ecologically because it means bodies of water freeze from the top down and not the bottom up. Because ice floats on the surface of lakes in the winter and the water beneath the ice remains liquid, fish and other animals keep from freezing.

Polar molecules and ions are soluble in water Water molecules gather closely around any substance that bears an electrical charge, whether that substance carries a full charge (ion) or a charge separation (polar molecule). For example, sucrose (table sugar) is composed of molecules that contain polar hydroxyl (OH) groups. A sugar crystal dissolves rapidly in water because water molecules can form hydrogen bonds with individual hydroxyl groups of the sucrose molecules. Therefore, sucrose is said to be soluble in water. Water is termed the solvent, and sugar is called the solute. Every time a sucrose molecule dissociates, or breaks away, from a solid sugar crystal, water molecules surround it in a cloud, forming a hydration shell that prevents it from associating with other sucrose molecules. Hydration shells also form around ions such as Na+ and Cl− (figure 2.15).

Water organizes nonpolar molecules Water molecules always tend to form the maximum possible num­ ber of hydrogen bonds. When nonpolar molecules such as oils, which do not form hydrogen bonds, are placed in water, the water molecules act to exclude them. The nonpolar molecules aggregate, or clump together, thus minimizing their disruption of the hydro­ gen bonding of water. In effect, they shrink from contact with wa­ ter, and for this reason they are referred to as hydrophobic (Greek hydros, “water,” and phobos, “fearing”). In contrast, polar mole­ cules, which readily form hydrogen bonds with water, are said to be hydrophilic (“water-loving”). The tendency of nonpolar molecules to aggregate in water is known as hydrophobic exclusion. By forcing the hydrophobic portions of molecules together, water can influence the shape of these molecules. This property is important to the structure of chapter

2 The Nature of Molecules and the Properties of Water 29

Learning Outcomes Review 2.5 δ−

δ− Water molecules

δ−

Na+

δ−

δ− Hydration shells

Water has a high specific heat so it does not change temperature rapidly, which helps living systems maintain a near-constant temperature. Water’s high heat of vaporization allows cooling by evaporation. Solid water is less dense than liquid water because the hydrogen bonds space the molecules farther apart. Polar molecules are soluble in a water solution, but water tends to exclude nonpolar molecules. Water dissociates to form H+ and OH–. ■■

Na+

How does the fact that ice floats affect life in a lake?

Cl− δ+ δ+ Cl− δ+

δ+

Learning Outcomes 1. Define acids, bases, and the pH scale. 2. Relate changes in pH to changes in [H +].

Figure 2.15 Why salt dissolves in water. When a crystal of table salt dissolves in water, individual Na+ and Cl− ions break away from the salt lattice and become surrounded by water molecules. Water molecules orient around Na+ so that their partial negative poles face toward the positive Na+; water molecules surrounding Cl− orient in the opposite way, with their partial positive poles facing the negative Cl−. Surrounded by hydration shells, Na+ and Cl− never reenter the salt lattice. proteins, DNA, and biological membranes. In fact, the interaction of nonpolar molecules and water is critical to living systems.

Water can form ions The covalent bond holding one hydrogen atom to the oxygen atom in a water molecule can be spontaneously broken. In pure water at 25°C, only 1 out of every 550 million water molecules undergoes this process. When it happens, a proton (hydrogen atom nucleus) dissociates from the molecule. Because the dissociated proton lacks the negatively charged electron it was sharing, its positive charge is no longer counterbalanced, and it becomes a hydrogen ion, H+. The rest of the dissociated water molecule, which has re­ tained the shared electron from the covalent bond, is negatively charged and forms a hydroxide ion, OH−. This process of sponta­ neous ion formation is called ionization:

water

and Bases

δ+

Salt crystal

H2O ⟶

2.6 Acids

OH− +

H+

hydroxide ion   hydrogen ion (proton)

At 25°C, 1 liter (L) of water contains one ten-millionth (or 10−7) mole of H+ ions. A mole (mol) is defined as the weight of a sub­ stance in grams that corresponds to the atomic masses of all of the atoms in a molecule of that substance. In the case of H+, the atomic mass is 1, and a mole of H+ ions would weigh 1 g. One mole of any substance always contains 6.02 × 1023 molecules of the substance. Therefore, the molar concentration of hydrogen ions in pure water, represented as [H+], is 10−7 mol/L. (In reality, the H+ usually associates with another water molecule to form a hydronium ion, H3O+.) 30 part I The Molecular Basis of Life

The concentration of hydrogen ions, and concurrently of hydroxide ions, in a solution is described by the terms acidity and basicity, respec­ tively. Pure water, having an [H+] of 10−7 mol/L, is considered to be neutral—that is, neither acidic nor basic. Recall that for every H+ ion formed when water dissociates, an OH− ion is also formed, meaning that the dissociation of water produces H+ and OH− in equal amounts.

The pH scale measures hydrogen ion concentration The pH scale (figure 2.16) is a more convenient way to express the hydrogen ion concentration of a solution. This scale defines pH, which stands for “power of hydrogen,” as the negative logarithm of the hydrogen ion concentration in the solution: pH = −log [H+] Because the logarithm of the hydrogen ion concentration is simply the exponent of the molar concentration of H+, the pH equals the exponent times −1. For water, therefore, an [H+] of 10−7 mol/L cor­ responds to a pH value of 7. This is the neutral point—a balance be­ tween H+ and OH−—on the pH scale. This balance occurs because the dissociation of water produces equal amounts of H+ and OH−. Note that, because the pH scale is logarithmic, a difference of 1 on the scale represents a 10-fold change in [H+]. A solution with a pH of 4 therefore has 10 times the [H+] of a solution with a pH of 5 and 100 times the [H+] of a solution with a pH of 6.

Acids Any substance that dissociates in water to increase the [H+] (and low­ er the pH) is called an acid. The stronger an acid is, the more hydro­ gen ions it produces and the lower its pH. For example, hydrochloric acid (HCl), which is abundant in your stomach, ionizes completely in water. A dilution of 10−1 mol/L of HCl dissociates to form 10−1 mol/L of H+, giving the ­solution a pH of 1. The pH of champagne, which bubbles because of the carbonic acid dissolved in it, is about 2.

pH Value

Examples of Solutions

100 10 −1

0 1

Hydrochloric acid

−2

10 10 −3

2 3

Stomach acid, lemon juice Vinegar, cola, beer

10 −4 10 −5

4

Tomatoes

5

Black coffee

10 −6

6

Urine

10 −7 10 −8

7

Pure water

Acidic

8

Seawater

−9

9

Baking soda

10 −10

10

Great Salt Lake

10 −11

11

Household ammonia

10 −12

12

10 −13

13

−14

14

10

10

Household bleach Basic

Sodium hydroxide

Figure 2.16 The pH scale. The pH value of a solution

indicates its concentration of hydrogen ions. Solutions with a pH less than 7 are acidic, whereas those with a pH greater than 7 are basic. The scale is logarithmic, which means that a pH change of 1 represents a 10-fold change in the concentration of hydrogen ions. Thus, lemon juice is 100 times more acidic than tomato juice, and seawater is 10 times more basic than pure water, which has a pH of 7.

Bases A substance that combines with H+ when dissolved in water, and thus lowers the [H+], is called a base. Therefore, basic (or alkaline) solutions have pH values above 7. Very strong bases, such as so­ dium hydroxide (NaOH), have pH values of 12 or more. Many common cleaning substances, such as ammonia and bleach, ac­ complish their action because of their high pH.

Buffers help stabilize pH The pH inside almost all living cells, and in the fluid surrounding cells in multicellular organisms, is fairly close to neutral, 7. Most of the enzymes in living systems are extremely sensitive to pH. Often even a small change in pH will alter their shape, thereby disrupting their activities. For this reason, it is important that a cell maintain a constant pH level. But the chemical reactions of life constantly produce acids and bases within cells. Furthermore, many animals eat substances that are acidic or basic. Cola drinks, for example, are moderately strong (although dilute) acidic solutions. Despite such variations in the concentrations of H+ and OH−, the pH of an organism is kept at a relatively constant level by buffers (figure 2.17). A buffer is a substance that resists changes in pH. Buffers act by releasing hydrogen ions when a base is added and absorbing hydrogen ions when acid is added, with the overall effect of keep­ ing [H+] relatively constant. Within organisms, most buffers consist of pairs of substanc­ es, one an acid and the other a base. The key buffer in human blood is an acid–base pair consisting of carbonic acid (acid) and bicar­ bonate (base). These two substances interact in a pair of reversible reactions. First, carbon dioxide (CO2) and H2O join to form

pH

Hydrogen Ion Concentration [H+]

9 8 7 6 5 4 3 2 1 0

Buffering range

0

1X

2X 3X 4X Amount of base added

5X

Figure 2.17 Buffers minimize changes in pH. Adding a base to a solution neutralizes some of the acid present, and so raises the pH. Thus, as the curve moves to the right, reflecting more and more base, it also rises to higher pH values. A buffer makes the curve rise or fall very slowly over a portion of the pH scale, called the “buffering range” of that buffer. carbonic acid (H2CO3), which in a second reaction dissociates to yield bicarbonate ion (HCO3−) and H+.

Data analysis If we call each step on the x-axis one

volume of base, how many volumes of base must be added to change the pH from 4 to 6?

If some acid or other substance adds H+ to the blood, the HCO3− acts as a base and removes the excess H+ by forming H2CO3. Similarly, if a basic substance removes H+ from the blood, H2CO3 dissociates, releasing more H+ into the blood. The forward and reverse reactions that interconvert H2CO3 and HCO3− thus stabilize the blood’s pH: −

+

Water (H2O) +

+ Carbon dioxide (CO2)

Carbonic acid (H2CO3)

+

Bicarbonate Hydrogen + ion ion (HCO3−) (H+)

The reaction of carbon dioxide and water to form carbonic acid is a crucial one because it permits carbon, essential to life, to enter water from the air. The Earth’s oceans are rich in carbon be­ cause of the reaction of carbon dioxide with water. In a condition called blood acidosis, human blood, which normally has a pH of about 7.4, drops to a pH of about 7.1. This condition is fatal if not treated immediately. The reverse condition, blood alkalosis, involves an increase in blood pH of a similar mag­ nitude and is just as serious.

Learning Outcomes Review 2.6

Acid solutions have a high [H+], and basic solutions have a low [H+] (and therefore a high [OH–]). The pH of a solution is the negative logarithm of its [H+]. Low pH values indicate acids, and high pH values indicate bases. Even small changes in pH can be harmful to life. Buffer systems in organisms help to maintain pH within a narrow range. ■■

A change of 2 pH units indicates what change in [H +]?

chapter

2 The Nature of Molecules and the Properties of Water 31

Chapter Review Virtualphoto/Getty Images

2.1 The Nature of Atoms All matter is composed of atoms (figure 2.3). Atomic structure includes a central nucleus and orbiting electrons. Electrically neutral atoms have the same number of protons as electrons. Atoms that gain or lose electrons are called ions. Elements are defined by the number of protons in the nucleus, the atomic number. Atomic mass is the sum of the mass of protons and neutrons. Isotopes are forms of a single element with different atomic mass due to different numbers of neutrons. Radioactive isotopes are unstable. Electrons determine the chemical behavior of atoms. The potential energy of electrons increases as distance from the nucleus increases. Electron orbitals are probability distributions. S orbitals are spherical; other orbitals have different shapes, such as the dumbbellshaped p orbitals. Atoms contain discrete energy levels. Energy levels correspond to quanta (singular, quantum) of energy, a “ladder” of energy levels that an electron may have. The loss of electrons from an atom is called oxidation. The gain of electrons is called reduction. Electrons can be transferred from one atom to another in coupled redox reactions.

2.2 Elements Found in Living Systems The periodic table displays elements according to atomic number and properties. Atoms tend to establish completely full outer energy levels (the octet rule). Elements with filled outermost orbitals are inert. There are 94 naturally occurring elements. Twelve of these elements are found in living organisms in greater than trace amounts: O, C, H, N, P, S, Na, K, Ca, Mg, Fe, and Cl. Compounds of carbon are called organic compounds. The majority of molecules in living systems are composed of C bound to H, O, and N.

2.3 The Nature of Chemical Bonds Molecules contain two or more atoms joined by chemical bonds. Compounds contain two or more different elements. Ionic bonds form crystals. Ions with opposite electrical charges form ionic bonds, such as NaCl (figure 2.9b). Covalent bonds build stable molecules. A molecule formed by a covalent bond is stable because it has no net charge, the octet rule is satisfied, and it has no unpaired electrons. Covalent bonds may be single, double, or triple, depending on the number of pairs of electrons shared. Nonpolar covalent bonds involve equal sharing of electrons between atoms. Polar covalent bonds involve unequal sharing of electrons. Chemical reactions alter bonds. Temperature, reactant concentration, and the presence of catalysts affect reaction rates. Most biological reactions are reversible, such as the conversion of carbon dioxide and water into carbohydrates.

32 part I The Molecular Basis of Life

2.4 Water: A Vital Compound Water’s structure facilitates hydrogen bonding. Hydrogen bonds are weak interactions between a partially positive H in one molecule and a partially negative O in another molecule (figure 2.11). Water molecules are cohesive. Cohesion is the tendency of water molecules to adhere to one another due to hydrogen bonding. The cohesion of water is responsible for its surface tension. Water molecules are adhesive. Adhesion occurs when water molecules adhere to other polar molecules. Capillary action results from water’s adhesion to the sides of narrow tubes, combined with its cohesion.

2.5 Properties of Water Water’s high specific heat helps maintain temperature. The hydrogen bonds in water act to increase specific heat. Organisms are mainly water, so they do not change temperature rapidly. Water’s high heat of vaporization facilitates cooling. Breaking hydrogen bonds to turn liquid water into vapor takes a lot of energy. Many organisms lose excess heat through evaporative cooling, such as sweating. Solid water is less dense than liquid water. Hydrogen bonds are spaced farther apart in the solid phase of water than in the liquid phase. As a result, ice floats. Polar molecules and ions are soluble in water. Water’s polarity makes it a good solvent for polar substances and ions. Polar molecules or portions of molecules are attracted to water (hydrophilic). Molecules that are nonpolar are repelled by water (hydrophobic). Water makes nonpolar molecules clump together. Water organizes nonpolar molecules. Nonpolar molecules will aggregate to avoid water. This maximizes the hydrogen bonds that water can make. This hydrophobic exclusion can affect the structure of DNA, proteins, and biological membranes. Water can form ions. Water dissociates into H+ and OH−. The concentration of H+, shown as [H+], in pure water is 10−7 mol/L.

2.6 Acids and Bases The pH scale measures hydrogen ion concentration (figure 2.16). pH is defined as the negative logarithm of [H+]. Pure water has a pH of 7. A difference of 1 pH unit means a 10-fold change in [H+]. Acids have a greater [H+] and therefore a lower pH; bases have a lower [H+] and therefore a higher pH. Buffers help stabilize pH. Carbon dioxide and water react reversibly to form carbonic acid. A buffer resists changes in pH by absorbing or releasing H+. The key buffer in the human blood is the carbonic acid/bicarbonate pair.

Visual Summary Chemistry of life

Organic = carbon compounds

Protons composed of

Neutrons Electrons

Important elements for life

Atoms

Molecules opposite charges

Carbon

Hydrogen

form Crystals

Water

Polar

Nitrogen

Dissociates into

Bonds Hydrogen bonds

hared electrons el shared Ionic

Oxygen

is

affect properties

Covalent unequal Polar

can form

ratio determines

H+

pH

equal Nonpolar

OH–

Cohesion

Adhesion

High specific head

Less dense solid High heat of vaporization

defined as pH = –log[H+] Solvent

>7

H+

H+ > OH–

Review Questions Virtualphoto/Getty Images

U N D E R S TA N D 1. The property that distinguishes an atom of one element (carbon, for example) from an atom of another element (oxygen, for example) is a. b. c. d.

the number of electrons. the number of protons. the number of neutrons. the combined number of protons and neutrons.

2. If an atom has one valence electron—that is, a single electron in its outer energy level—it will most likely form a. b. c. d.

one polar, covalent bond. two nonpolar, covalent bonds. two covalent bonds. an ionic bond.

3. An atom with a net positive charge must have more a. b. c. d.

protons than neutrons. protons than electrons. electrons than neutrons. electrons than protons.

4. The isotopes carbon-12 and carbon-14 differ in a. b. c. d.

the number of neutrons. the number of protons. the number of electrons. Both b and c are correct.

5. Which of the following is NOT a property of the elements most commonly found in living organisms? a. b. c. d.

The elements have a low atomic mass. The elements have an atomic number less than 21. The elements possess eight electrons in their outer energy level. The elements are lacking one or more electrons from their outer energy level.

6. Ionic bonds arise from a. b. c. d.

shared valence electrons. attractions between valence electrons. charge attractions between valence electrons. attractions between ions of opposite charge.

chapter

2 The Nature of Molecules and the Properties of Water 33

7. A solution with a high concentration of hydrogen ions a. b. c. d.

is called a base. is called an acid. has a high pH. Both b and c are correct.

A P P LY 1. Using the periodic table on page 23, which of the following atoms would you predict should form a positively charged ion (cation)? a. b. c. d.

Fluorine (F) Neon (Ne) Potassium (K) Sulfur (S)

2. Refer to the element pictured. How many covalent bonds could this atom form? a. b. c. d.

Two Three Four None

3. A molecule with polar covalent bonds would a. b. c. d.

be soluble in water. not be soluble in water. contain atoms with very similar electronegativity. Both b and c are correct.

4. Hydrogen bonds are formed a. b. c. d.

between any molecules that contain hydrogen. only between water molecules. when hydrogen is part of a polar bond. when two atoms of hydrogen share an electron.

34 part I The Molecular Basis of Life

5. If you shake a bottle of oil and vinegar then let it sit, it will separate into two phases because a. b. c. d.

the nonpolar oil is soluble in water. water can form hydrogen bonds with the oil. polar oil is not soluble in water. nonpolar oil is not soluble in water.

6. The decay of radioactive isotopes involves changes to the nucleus of atoms. Explain how this differs from the changes in atoms that occur during chemical reactions.

SYNTHESIZE 1. Based on their position in the periodic table, how would you predict bromine (Br) and lithium (Li) would react? What kind of compound might be formed, and what kinds of bonds would you expect? 2. A popular theme in science fiction literature has been the idea of silicon-based life-forms in contrast to our carbon-based life. Evaluate the possibility of silicon-based life based on the chemical structure and potential for chemical bonding of a silicon atom. 3. Efforts by NASA to search for signs of life on Mars have focused on the search for evidence of liquid water rather than looking directly for biological organisms (living or fossilized). Use your knowledge of the influence of water on life on Earth to construct an argument justifying this approach.

PartCHAPTER VIII Genetic and Molecular Biology

3

The Chemical Building Blocks of Life Chapter Contents 3.1 Carbon: The Framework of Biological Molecules 3.2 Carbohydrates: Energy Storage and Structural Molecules 3.3 Nucleic Acids: Information Molecules 3.4 Proteins: Molecules with Diverse Structures and Functions 3.5 Lipids: Hydrophobic Molecules

Deco/Alamy Stock Photo

Introduction

Visual Outline Macromolecules

carbon based

Polymers

Organic molecules

contain

Functional groups

Nonpolymers

form byy dehydration reactions dra rea Carbohydrates

Nucleic acids

types Simple sugars

Complex

monomers are

CH2OH 5 C O H H C C H 1 4 OH C C OH OH 3 2 H

Nucleotides Simple sugars

OH

DNA: Molekuul/SPL/age fotostock

Lipids

monomers are

types

Amino acids

monomers are

H

Proteins

Triglycerides

Phospholipids

made of

made of

Glycerol & 3 fatty acids

Glycerol, 2 fatty acids & polar head

Despite the complexity and diversity of living systems, all organisms share the same basic chemistry. In chapter 2, we saw how this involves a limited number of elements; now we will see that organisms consist of a few basic kinds of molecules. These molecules are all built on a carbon framework, making them organic molecules. These are all synthesized by living things, and are called macromolecules because they can be very large molecules, like the DNA and proteins shown interacting in the photo. We recognize four classes of biological macromolecules: carbohydrates, nucleic acids, proteins, and lipids. We take the existence of these classes of macromolecules for granted now, but as late as the 19th century many theories of “vital forces” were associated with living systems. One such theory held that cells contained a substance, p ­ rotoplasm, that was responsible for the chemical

reactions in living systems. Any disruption of cells was thought to disturb the p ­ rotoplasm. Such a view makes studying the chemical reactions of cells in a test tube (in vitro) impossible. The ­demonstration of fermentation in a cell-free ­system marked the beginning of modern biochemistry (figure 3.1). This approach involves ­s tudying biological molecules outside of cells to infer their role inside cells. Because these biological macromolecules all involve carbon-containing compounds, we begin with a brief summary of carbon and its chemistry.

3.1 Carbon:

The Framework of Biological Molecules

Learning Outcomes 1. Describe the relationship between functional groups and macromolecules. 2. Recognize the different kinds of isomers. 3. List the different kinds of biological macromolecules.

In chapter 2, we reviewed the basics of atomic structure and chemical bonding. Biological systems obey all the laws of chemistry. Thus, chemistry forms the basis of living systems. The framework of biological molecules consists predominantly of carbon atoms bonded to other carbon atoms or to atoms of hydrogen, oxygen, nitrogen, sulfur, or phosphorus. Because carbon atoms can form up to four covalent bonds, molecules containing carbon can form straight chains, branches, or even rings, balls, tubes, and coils. Molecules consisting only of carbon and hydrogen are called hydrocarbons. Because the oxidation of hydrocarbon compounds results in a net release of energy, hydrocarbons make good fuels. Gasoline, for example, is rich in hydrocarbons, and propane gas, another hydrocarbon, consists of a chain of three carbon atoms, with eight hydrogen atoms bound to it. The chemical formula for propane is C3H8. Its structural formula is H H H H— C— C— C—H H H H

Propane structural formula

Theoretically speaking, the length of a chain of carbon atoms is unlimited. As described in the rest of this chapter, the four main types of biological molecules often consist of huge chains of carbon-containing compounds.

SCIENTIFIC THINKING Hypothesis: Chemical reactions, such as the fermentation reaction in yeast, are controlled by enzymes and do not require living cells. Prediction: If yeast cells are broken open, these enzymes should function outside of the cell. Test: Yeast is mixed with quartz sand and diatomaceous earth and then ground in a mortar and pestle. The resulting paste is wrapped in canvas and subjected to 400–500 atm pressure in a press. Fermentable and nonfermentable substrates are added to the resulting fluid, with fermentation being measured by the production of CO2. Yeast

Quartz sand

Diatomaceous earth

Grind in mortar/pestle.

400–500 atm pressure

Cane sugar

Glucose

Lactose, mannose

Wrap in canvas and apply pressure in a press.

Result: When a fermentable substrate (cane sugar, glucose) is used, CO2 is produced; when a nonfermentable substrate (lactose, mannose) is used, no CO2 is produced. In addition, visual inspection of the fluid shows no visible yeast cells. Conclusion: The hypothesis is supported. The fermentation reaction can occur in the absence of live yeast. Historical Significance: Although this is not precisely the intent of the original experiment, it represents the first use of a cell-free system. Such systems allow for the study of biochemical reactions in vitro and the purification of proteins involved. We now know that the “fermentation reaction” is actually a complex series of reactions. Would such a series of reactions be your first choice for this kind of demonstration?

Figure 3.1 The demonstration of cell-free fermentation. The German chemist Eduard Buchner’s (1860–1917) demonstration of

fermentation by fluid produced from yeast, but not containing any live cells, both argued against the protoplasm theory and provided a method for future biochemists to examine the chemistry of life outside of cells. 36 part I The Molecular Basis of Life

Functional groups account for differences in molecular properties Carbon and hydrogen atoms both have very similar electronegativities. Electrons in C—C and C—H bonds are therefore evenly distributed, with no significant differences in charge over the molecular surface. For this reason, hydrocarbons are nonpolar. Most Functional Group

Structural Formula

Hydroxyl

Found In

Example

OH

H

H

C

C

H

OH

carbohydrates, proteins, nucleic acids, lipids

H

carbohydrates, nucleic acids

H H Ethanol

O

Carbonyl

H

C

C

C

H

C

H

proteins, lipids

OH H Acetic acid

H N

O C

C

OH

Amino

O

H Acetaldehyde O

Carboxyl

H

HO

O

H

C

C

H

H

proteins, nucleic acids

N H

CH3 Alanine COOH

Sulfhydryl

S

H

H

S

CH2

C

Phosphate

O– H

P

O

O

C

C

C

H

H

H

X

X

C O

O

Organic molecules having the same molecular or empirical formula can exist in different forms called isomers. If there are differences in the actual structure of their carbon skeleton, we call them structural isomers. In section 3.2, you will see that glucose and fructose are structural isomers of C6H12O6. Another form of isomers, called stereoisomers, have the same carbon skeleton but differ in how the groups attached to this skeleton are arranged in space. Enzymes in biological systems usually recognize only a single, specific stereoisomer. A subcategory of stereoisomers, called enan­tiomers, are actually mirror images of each other. A molecule that has mirror-image versions is called a chiral molecule. When carbon is bound to four different molecules, this inherent asymmetry exists (figure 3.3). W

Cysteine OH OH H

Isomers have the same molecular formulas but different structures

proteins

H

NH2

O–

biological molecules produced by cells, however, also contain other atoms. Because these other atoms frequently have different electronegativities (see table 2.2), molecules containing them exhibit regions of partial positive or negative charge. They are polar. These molecules can be thought of as a C—H core to which specific molecular groups, called ­functional groups, are attached. One such common functional group is —OH, called a hydroxyl group. Functional groups have definite chemical properties that they retain no matter where they occur. Both the hydroxyl and carbonyl (C==O) groups, for example, are polar because of the electronegativity of the oxygen atoms (see chapter 2). Other common functional groups are the acidic carboxyl (COOH), the phosphate (PO4−), and the basic amino (NH2) groups. Many of these functional groups can also participate in hydrogen bonding. Hydrogen bond donors and acceptors can be predicted based on their electronegativities, shown in table 2.2. Figure 3.2 illustrates these biologically important functional groups and lists the macromolecules in which they are found.

P

O–

nucleic acids

Z

W C

Y

Y

Z

O–

Glycerol phosphate H Methyl

C H

H

HO

O

H

C

C

NH2

H

C

H

proteins, nucleic acids

H Alanine

Figure 3.2 The primary functional chemical groups. 

These groups tend to act as units during chemical reactions and give specific chemical properties to the molecules that possess them. Amino groups, for example, make a molecule more basic, and carboxyl groups make a molecule more acidic. These functional groups are also not limited to the examples in the “Found in” column but are widely distributed in biological molecules.

Mirror

Figure 3.3 Chiral molecules. When carbon is bound to

four different groups, the resulting molecule is said to be chiral (from Greek cheir, meaning “hand”). A chiral molecule will have stereoisomers that are mirror images. The two molecules shown have the same four groups but cannot be superimposed, much like your two hands, which cannot be superimposed but must be flipped to match. These types of stereo­iso­mers are called enantiomers. chapter

3 The Chemical Building Blocks of Life 37

Cellular Structure

Polymer

Monomer

Carbohydrate

CH2OH O

H HO

Starch grains in a chloroplast

Starch

H

H OH

H

H

OH

OH

Monosaccharide

P

P

Nucleic Acid

G

P

P

Nitrogenous base

P

T

T A

G A

C

P

P

P P

C

P

A P

P

A

T

C

P

Protein

Chromosome

Nucleotide

Val Val

Ser

Polypeptide

5-carbon sugar OH

DNA strand

Ala

Intermediate filament

P

P

P

O

Phosphate group

G

Ala

CH3

H N H

C

C

H

O

OH

Amino acid

Lipid

O H H H H H H H H H H H HO C C C C C C C C C C C C H H H H H H H H H H H H

Adipose cell with fat droplets

Triglyceride

Fatty acid

Figure 3.4 Polymer macromolecules. The four major biological macromolecules are shown. Carbohydrates, nucleic acids, and proteins all form polymers and are shown with the monomers used to make them. Lipids do not fit this simple monomer–polymer relationship. The triglyceride shown is constructed from glycerol and fatty acids. All four types of macromolecules are also shown in their cellular context.

38 part I The Molecular Basis of Life

TA B L E 3 .1

Macromolecules

Macromolecule

Subunit

Function

Example

CA RBO H Y D R ATE S Starch, glycogen

Glucose

Energy storage

Potatoes

Cellulose

Glucose

Structural support in plant cell walls

Paper; strings of celery

Chitin

Modified glucose

Structural support

Crab shells

DNA

Nucleotides

Encodes genes

Chromosomes

RNA

Nucleotides

Needed for gene expression

Messenger RNA

NUCLEIC ACIDS

PROTEINS Functional

Amino acids

Catalysis; transport

Hemoglobin

Structural

Amino acids

Support

Hair; silk

LIPIDS Triglycerides (animal fat, oils)

Glycerol and three fatty acids

Energy storage

Butter; corn oil; soap

Phospholipids

Glycerol, two fatty acids, phosphate, and polar R groups

Cell membranes

Phosphatidylcholine

Prostaglandins

Five-carbon rings with two nonpolar tails

Chemical messengers

Prostaglandin E (PGE)

Steroids

Four fused carbon rings

Membranes; hormones

Cholesterol; estrogen

Terpenes

Long carbon chains

Pigments; structural support

Carotene; rubber

Chiral compounds are characterized by their effect on polarized light. Polarized light has a single plane, and chiral molecules rotate this plane to either the right (Latin, dextro) or the left (Latin, levo). We therefore call the two chiral forms D for dextrorotatory and L for levorotatory. Living systems tend to produce only a single enantiomer of the two possible forms; for example, in most organisms we find primarily d-sugars and l‑amino acids.

Biological macromolecules include carbohydrates, nucleic acids, proteins, and lipids Remember that biological macromolecules are traditionally grouped into carbohydrates, nucleic acids, proteins, and lipids (table 3.1). In many cases, these macromolecules are polymers. A polymer is a long molecule built by linking together a large number of small, similar chemical subunits called monomers. They are like railroad cars coupled to form a train. The nature of a polymer is determined by its constituent monomers. For example, the complex carbohydrate starch is a polymer composed of subunits of the simple sugar glucose. Nucleic acids (DNA and RNA) are polymers of nucleotides, and proteins are polymers of amino acids (figure 3.4). These long chains are built via chemical reactions termed dehydration reactions and are broken down by hydrolysis reactions. Lipids are macromolecules, but they really don’t follow the monomer–polymer relationship. However, lipids are formed through dehydration reactions, which link the fatty acids to glycerol.

The dehydration reaction Despite the differences between monomers of these major polymers, the basic chemistry of their synthesis is similar: to form a covalent bond between two monomers, an —OH group is removed from one monomer, and a hydrogen atom (H) is removed from the other (figure 3.5a). This reaction is the same for joining nucleotides when synthesizing DNA or joining glucose units together to make starch. This reaction is also used to link fatty acids to glycerol in lipids. This chemical reaction is called condensation, or a dehydration reaction, because the removal of —OH and —H is the same as the removal of a molecule of water (H2O). For every subunit added to a macromolecule, one water molecule is removed. These and other biochemical reactions require that the reacting substances are held close together and that the correct chemical H2O HO

H

HO

HO

H

H2O HO

H

HO

a. Dehydration reaction

H

H

HO

H

b. Hydrolysis reaction

Figure 3.5 Making and breaking macromolecules. 

a. Biological macromolecules are polymers formed by linking monomers together through dehydration reactions. This process releases a water molecule for every bond formed. b. Breaking the bond between subunits involves hydrolysis, which reverses the loss of a water molecule by dehydration. chapter

3 The Chemical Building Blocks of Life 39

Monosaccharides are simple sugars

bonds are stressed and broken. This process of positioning and stressing, termed catalysis, is carried out within cells by enzymes.

Carbohydrates are a loosely defined group of molecules that all contain carbon, hydrogen, and oxygen in the molar ratio 1:2:1. Their empirical formula (which lists the number of atoms in the molecule with subscripts) is (CH2O)n , where n is the number of carbon atoms. Because they contain many carbon–hydrogen (C—H) bonds, which release energy when they are rearranged, carbohydrates are well suited for energy storage. Sugars are among the most important energy-storage molecules, and they exist in several different forms. The simplest of the carbohydrates are the ­monosaccharides (Greek mono, “single,” and Latin saccharum, “sugar”). Simple sugars contain as few as three carbon atoms, but those that play the central role in energy storage have six (figure 3.6). The empirical formula of 6-carbon sugars is:

The hydrolysis reaction Cells disassemble polymers into their constituent monomers by reversing the dehydration reaction—a molecule of water is added instead of removed (figure 3.5b). In this reaction, called hydrolysis, a hydrogen atom is attached to one subunit and a ­hydroxyl group to the other, breaking the covalent bond joining the subunits. When you eat a potato, which contains starch (see section  3.2), your body breaks the starch down into glucose units by ­hydrolysis. The potato plant built the starch molecules originally by dehydration reactions.

Learning Outcomes Review 3.1

Functional groups account for differences in chemical properties in organic molecules. Isomers are compounds with the same empirical formula but different structures. This difference may affect biological function. Macromolecules are polymers consisting of long chains of similar subunits that are joined by dehydration reactions and are broken down by hydrolysis reactions. ■■

C6H12O6     or     (CH2O)6 Six-carbon sugars can exist in a straight-chain form, but dissolved in water (an aqueous environment) they almost always form rings. The most important 6-carbon sugar used for energy storage is glucose, which appeared in the photosynthesis reactions in chapter 2. Glucose has seven energy-storing C—H bonds (figure 3.7). Depending on the o­ rientation of the carbonyl group (C=O) when the ring is closed, glucose can exist in two different forms: alpha (α) or beta (β). This is significant, as we will see, when glucose is used as a monomer to form polymers.

What is the relationship between dehydration and hydrolysis?

Sugar isomers have structural differences

3.2  Carbohydrates:

Energy Storage and Structural Molecules

Glucose is not the only sugar with the formula C6H12O6. Both structural isomers and stereoisomers of this simple 6-carbon skeleton exist in nature. Fructose is a structural isomer that differs in the position of the carbonyl carbon (C=O); galactose is a ste­ reo­iso­mer that differs in the position of —OH and —H groups relative to the ring (figure 3.8). These differences often account for substantial functional differences between the isomers. Your taste buds can discern them: fructose tastes much sweeter than glucose, despite the fact that both sugars have identical chemical composition. Enzymes that act on different sugars can distinguish both the structural and stereoisomers of this basic 6-carbon

Learning Outcomes 1. Describe the structure of simple sugars with three to six carbons. 2. Relate the structure of polysaccharides to their functions.

3-carbon Sugar H

O 1

H H

2 3

5-carbon Sugars 5

C C

5

CH2OH

C

O OH OH

H Glyceraldehyde

4

H

H

H

OH

OH

3

H 2

Ribose

6

CH2OH O

OH 1

6-carbon Sugars

4

H

OH

H

H

OH

H

3

1

H 2

Deoxyribose

H 4

HO

6

CH2OH 5

H OH 3

H

O

2

OH

Glucose

O

H

H

6

CH2OH

1

5

OH

HO

H 4

OH

H 2

OH CH OH 2 3

H

Fructose

1

OH 4

H

CH2OH 5

H OH 3

H

O

1

H

H 2

OH

Galactose

Figure 3.6 Monosaccharides. Monosaccharides, or simple sugars, can contain as few as three carbon atoms and are often used as building blocks to form larger molecules. The 5-carbon sugars ribose and deoxyribose are components of nucleic acids (see figure 3.16). 40 part I The Molecular Basis of Life

OH

skeleton. The different stereoisomers of glucose are also important in the polymers that can be made using glucose as a monomer, as you will see later in this section.

CH2OH

5

H O 1

H HO H

2 3 4

C C C

C

OH H

H

OH

5

H

H

6

C

C

OH

C

OH

OH

4

3

C

OH

H

C H OH C

4

H O H C

3

O 1

O

H

OH

H C

H C 1

2

OH

Disaccharides serve as transport molecules in plants and provide nutrition in animals

α-glucose or β-glucose

C 2

C H OH C

H

Most organisms transport sugars within their bodies. In humans, the glucose that circulates in C O H OH the blood does so as a simple monosaccharide. In H C OH 6 C C H plants and many other organisms, however, gluOH H H 4 1 cose is converted into a transport form before it is OH H C C 3 2 moved from place to place within the organism. H OH In such a form, it is less readily metabolized during transport. Figure 3.7 Structure of the glucose molecule. Glucose is a linear, 6-carbon Transport forms of sugars are commonly molecule that forms a six-membered ring in solution. Ring closure occurs such that two made by linking two monosaccharides together forms can result: α-glucose and β-glucose. These structures differ only in the position of to form a disaccharide (Greek di, “two”). Disacthe —OH bound to carbon 1. The structure of the ring can be represented in many ways; charides serve as effective reservoirs of glucose shown here are the most common, with the carbons conventionally numbered so that the because the enzymes that normally use glucose forms can be compared easily. The heavy lines in the ring structures represent portions of in the organism cannot break the bond linking the molecule that are projecting out of the page toward you. the two monosaccharide subunits. Enzymes that can do so are typically present only in the tissue that uses glucose. H H H Transport forms differ depending on which monosacchaC O H C OH C O rides are linked to form the disaccharide. Glucose forms transport Structural H C StereoO H C OH C disaccharides with itself and with many other monosaccharides, OH isomer isomer including fructose and galactose. When glucose forms a disacchaHO H HO H HO H C C C ride with the structural isomer fructose, the resulting disaccharide HO H C H C OH H C OH is sucrose, or table sugar (­figure 3.9a). Sucrose is the form most plants use to transport glucose and is the sugar that most humans H C OH H C OH H C OH and other animals eat. Sugarcane and sugar beets are rich in H C OH H C OH H C OH sucrose. When glucose is linked to the stereoisomer galactose, the reH H H sulting disaccharide is ­lactose, or milk sugar. Many mammals supFructose Glucose Galactose ply energy to their young in the form of lactose. Adults often have greatly reduced levels of lactase, the enzyme required to cleave Figure 3.8 Isomers and stereoisomers. Glucose, fructose, lactose into its two monosaccharide components, and thus they and galactose are isomers with the empirical formula C6H12O6. A cannot metabolize lactose efficiently. This can result in lactose instructural isomer of glucose, such as fructose, has identical chemical tolerance in humans. Most of the energy that is channeled into lacgroups bonded to different carbon atoms. Notice that this results in a tose production is therefore reserved for offspring. For this reason, five-membered ring in solution (see figure 3.6). A stereoisomer of lactose as an energy source is primarily for offspring in mammals. glucose, such as galactose, has identical chemical groups bonded to the H

5

H

CH2OH

OH

5

same carbon atoms but in different orientations (the —OH at carbon 4). CH2OH H HO

CH2OH O

H OH

H

OH H α-glucose

a.

H OH

CH2OH O

+ HO

H

H OH

OH H Fructose

H

CH2OH

HO H 2O

H OH H

H

O

H O

OH

CH2OH

CH2OH

CH2OH O

H

H

OH CH OH 2

OH

H

H HO

O H OH H

Sucrose

H

H

H O

O H OH

H OH

H

OH

H

OH

Maltose

b.

Figure 3.9 How disaccharides form. Some disaccharides are used to transport glucose from one part of an organism’s body to another; one example is sucrose (a), which is found in sugarcane. Other disaccharides, such as maltose (b), are used in grain for storage. chapter

3 The Chemical Building Blocks of Life 41

Polysaccharides provide energy storage and structural components

c­ ontaining branched amylose chains. Glycogen has a much longer average chain length and more branches than plant starch (figure 3.10c).

Polysaccharides are longer polymers made up of monosaccharides that have been joined through dehydration reactions. Starch, a storage polysaccharide, consists entirely of α-­glucose molecules linked in long chains. Cellulose, a structural polysaccharide, also consists of glucose molecules linked in chains, but these molecules are β-glucose. Because starch is built from α-glucose we call the linkages α linkages; cellulose has β linkages.

Cellulose Although some chains of sugars store energy, others serve as structural material for cells. For two glucose molecules to link together, the glucose subunits must be of the same form. Cellulose is a polymer of β-glucose (figure 3.11). The bonds ­between adjacent glucose molecules still exist between the C-1 of the first glucose and the C-4 of the next glucose, but these are β-(1  4) linkages. The properties of a chain of glucose molecules consisting of all β-glucose are very different from those of starch. These long, unbranched β-linked chains make tough fibers. Cellulose is the chief component of plant cell walls (see figure 3.11b). It is chemically similar to amylose, with one important difference: the starchhydrolyzing enzymes that occur in most organisms cannot break the bond between two β-glucose units because they only recognize α linkages. Because cellulose cannot be broken down readily by most animals, it works well as a biological structural material. But some animals, such as cows, are able to utilize cellulose aided by symbiotic bacteria and protists in their digestive tracts. These organisms provide the necessary enzyme to cleave the β-(1  4) linkages, releasing glucose for further metabolism.

Starches and glycogen Organisms store the metabolic energy contained in monosaccharides by converting them into disaccharides, such as ­ maltose (figure 3.9b). These are then linked together into the insoluble polysaccharides called starches. These polysaccharides differ mainly in how the polymers branch. The starch with the simplest structure is amylose. It is composed of many hundreds of α-glucose molecules linked together in long, unbranched chains. Each linkage occurs between the carbon 1 (C-1) of one glucose molecule and the C-4 of another, making them α-(1  4) linkages (­figure 3.10a). The long chains of amylose tend to coil up in water, a property that renders amylose insoluble. Potato starch is about 20% amylose (­figure 3.10b). Most plant starch, including the remaining 80% of potato starch, is a somewhat more complicated variant of amylose called amylopectin. Pectins are branched polysaccharides with the branches occurring due to bonds between the C-1 of one molecule and the C-6 of another [α-(1  6) linkages]. These short amylose branches consist of 20 to 30 glucose subunits (figure 3.10b). The comparable molecule to starch in animals is glycogen. Like amylopectin, glycogen is an insoluble polysaccharide CH2OH H 4

HO

CH2OH O

H OH

H

H

H

1

CH2OH O

OH

H OH α-glucose

OH

Chitin, the structural material found in arthropods and many fungi, is a polymer of N-acetylglucosamine, a nitrogen-containing derivative of glucose. When cross-linked by proteins, it forms a tough, resistant surface material that serves as the hard exoskeleton of insects and crustaceans (figure 3.12; see chapter  33). Few

CH2OH O

H

Chitin

OH

H

O

H

H

OH

H OH α-1→4 linkages

O

H O

OH

H

H

OH

H

Amylose

CH2OH H

CH2OH H

O H OH

H

H

OH

O H OH H

a.

H

H

H O

H O

b.

+

Amylopectin

7.5 μm

α-1→6 linkage

CH2 O H OH

H OH α-1→4 linkage

H

H OH Glycogen

c.

3.3 μm

Figure 3.10 Polymers of glucose: Starch and glycogen. a. Starch chains consist of polymers of α-glucose subunits joined by

α-(1  4) glycosidic linkages. These chains can be branched by forming similar α-(1  6) glycosidic bonds. These storage polymers then differ primarily in their degree of branching. b. Starch is found in plants and is composed of amylose and amylopectin, which are unbranched and branched, respectively. The branched form is insoluble and forms starch granules in plant cells. c. Glycogen is found in animal cells and is highly branched and also insoluble, forming glycogen granules. (b) Asa Thoresen/Science Source; (c) Mike Rosecope/Shutterstock 42 part I The Molecular Basis of Life

CH2OH H 4

HO

CH2OH O

H OH H

H

OH

H

1

H

OH

O

O H OH H

H

O H

H

OH

OH H

H O

OH

β-glucose

H

CH2OH H

H O

CH2OH

O H OH

H

H

OH

O H

β-1→4 linkages

a.

b.

500 μm

Figure 3.11 Polymers of glucose: Cellulose. Starch chains consist of α-glucose subunits, and cellulose chains consist of β-glucose subunits. a. Thus the bonds between adjacent glucose molecules in cellulose are β-(1  4) glycosidic linkages. b. Cellulose is unbranched and forms long fibers. Cellulose fibers can be very strong and are quite resistant to metabolic breakdown, which is one reason wood is such a good building material. (b) Martin Kreutz/age fotostock

3.3  Nucleic

Acids: Information Molecules

Learning Outcomes Figure 3.12 Chitin. Chitin is the principal structural element in the external skeletons of many arthropods, such as this lobster. OAR/National Undersea Research Program (NURP)

organisms are able to digest chitin, but most possess a chitinase enzyme, probably to protect against fungi.

Learning Outcomes Review 3.2

Monosaccharides have three to six or more carbon atoms typically arranged in a ring form. Disaccharides consist of two linked monosaccharides; polysaccharides are long chains of monosaccharides. Structural differences between sugar isomers can lead to functional differences. Starches are branched polymers of α-glucose used for energy storage. Cellulose in plants consists of unbranched chains of β-glucose that are not easily digested. ■■

How do the structures of starch, glycogen, and cellulose affect their function?

1. 2. 3. 4.

Describe the structure of nucleotides. Contrast the structures of DNA and RNA. Discuss the functions of DNA and RNA. Recognize other nucleotides involved in energy metabolism.

The biochemical activity of a cell depends on production of a large number of proteins, each with a specific sequence. The information necessary to produce the correct proteins is passed through generations of organisms, even though the proteins themselves are not inherited. Nucleic acids carry information inside cells, just as hard drives contain the information in a computer or the GPS system in a car displays information needed by travelers. Two main varieties of nucleic acids are deoxyribonucleic acid (DNA; figure 3.13) and r­ ibonucleic acid (RNA). Genetic information is stored in DNA, while RNA plays a variety of roles in the cell, including as short-lived copies of genetic information used to direct the synthesis of proteins (as discussed in detail in chapter 15). Unique among macromolecules, nucleic acids are able to serve as templates for producing precise chapter

3 The Chemical Building Blocks of Life 43

Nitrogenous base 7N

O P

9

O

6

N

8

Phosphate group

−O

NH2 5

N

4

N

1 2

3

CH2

5′

O−

O 1′

4′ 3′

2′

OH Sugar

OH in RNA H in DNA

Figure 3.14 Structure of a nucleotide. The nucleotide

a.

2 nm

b.

subunits of DNA and RNA are made up of three elements: a 5-­carbon sugar (ribose or deoxyribose), an organic nitrogenous base (adenine is shown here), and a phosphate group. Notice that all the numbers on the sugar are given as “primes” (1´, 2´, etc.) to distinguish them from the numbering on the rings of the bases.

Figure 3.13 Images of DNA. a. A scanning-tunneling

micrograph of DNA (false color; 2,000,000×) showing approximately three turns of the DNA double helix. b. A space-filling model for comparison to the image of actual DNA in (a). (a) Driscoll, Youngquist &

Baldeschwieler, California Institute of Technology/Science Source; (b) Molekuul/ SPL/age fotostock

copies of themselves. This characteristic allows genetic information to be preserved during cell division and during the reproduction of organisms. Unlike DNA, which functions to store information, RNA has a variety of roles in the cell. It can carry genetic information, it is an integral part of the machinery for protein synthesis, and it can also function to regulate the process of gene expression. As a carrier of information, the form of RNA called messenger RNA (mRNA) consists of transcribed single-stranded copies of portions of the DNA. These transcripts serve as blueprints specifying the amino acid sequences of proteins. This process will be described in detail in chapter 15.

Nucleic acid polymers are polynucleotides Nucleic acids consist of long polymers of repeating subunits called nucleotides. Each nucleotide consists of three components: a 5-carbon sugar; a phosphate (—PO4−) group; and an organic nitrogen-containing, or nitrogenous, base. In DNA, the sugar is deoxyribose and in RNA it is ribose (figure 3.14). Nucleotides have five types of nitrogenous bases (figure 3.15). Two of these are large, double-ring molecules called purines that are each found in both DNA and RNA; the two purines are adenine (A) and guanine (G). The other three bases are single-ring molecules called pyrimidines that include cytosine (C, in both DNA and RNA), thymine (T, in DNA only), and uracil (U, in RNA only). Polynucleotides are formed by joining the phosphate of one nucleotide to a hydroxyl group on the sugar of another nucleotide 44 part I The Molecular Basis of Life

in a dehydration reaction. We call this a phosphodiester bond because the two sugars are linked by ester bonds to a phosphate. A nucleic acid, then, is simply a chain of 5-carbon sugars linked together by phosphodiester bonds with a nitrogenous base protruding from each sugar (figure 3.16). These polynucleotide chains have polarity, meaning the two ends are different. One end has a phosphate and the other an —OH from a sugar. We refer to these as the 5´ (“five-prime,” —PO4−) end and the 3´ (“three-prime,” —OH) end, based on the carbon numbering of the sugar (see figure 3.16).

DNA stores genetic information Organisms use sequences of nucleotides in DNA to encode the information specifying the amino acid sequences of their proteins. This method of encoding information is very similar to the way in which sequences of letters encode information in a sentence. A sentence written in English consists of a combination of the 26 different letters of the alphabet in a certain order; the code of a DNA molecule consists of different combinations of the four types of nucleotides in specific sequences, such as CGCTTACG. DNA molecules in organisms exist as two polynucleotide chains wrapped about each other in a long linear molecule in eukaryotes, and a circular molecule in most prokaryotes. The two strands of a DNA polymer wind around each other like the outside and inside rails of a spiral staircase. Such a spiral shape is called a helix, and a helix composed of two chains is called a double helix. Each step of DNA’s helical staircase is composed of a base-pair. The pair consists of a base in one chain attracted by hydrogen bonds to a base opposite it on the other chain (­figure 3.17). The base-pairing rules arise from the most stable hydrogen bonding configurations between the bases: adenine pairs with

Purines

H

C

N C C

N

N C

C H

N

structure of DNA and how it interacts with RNA in the production of proteins are presented in chapters 14 and 15. In eukaryotic organisms, the DNA is further complexed with protein to form structures we call chromosomes. This actually forms a higher-order structure that affects the function of DNA as it is involved in the control of gene expression (see chapter 16).

O

NH2 H

C

N C C

N

H

N C

C

NH2

N

H

H Adenine (both DNA and RNA)

Guanine (both DNA and RNA)

RNA has many roles in a cell

O

O

NH2 Pyrimidines

RNA is similar to DNA, but with two major chemical differences. First, RNA molecules contain ribose H C H C H C C O C O C O sugars, in which the C-2 is bonded to a hydroxyl N N N group. (In DNA, a hydrogen atom replaces this H H H hydroxyl group.) Second, RNA molecules use uracil Uracil Cytosine Thymine in place of thymine. Uracil has a similar structure (RNA only) (both DNA and RNA) (DNA only) to thymine, except that one of its carbons lacks a Figure 3.15 The organic nitrogenous bases are either purines (2 rings) methyl (—CH3) group. RNA is produced by transcription (copying) or pyrimidines (1 ring). The base thymine is found in DNA, and replaced from DNA, and is usually single-stranded (fig-­ by uracil in RNA. ure 3.18). The role of RNA in cells is quite varied: it carries information in the form of mRNA, it is part of the ribothymine (in DNA) or with uracil (in RNA), and cytosine pairs some, in the form of ribosomal RNA (rRNA), and it carries with guanine. The bases that participate in base-pairing are said to amino acids in the form of transfer RNA (tRNA). There has be complementary to each other. Additional details of the been a revolution of late in how we view RNA since it has been found to function as an enzyme, and other forms of RNA are involved in regulating gene expression (explored in more detail in 5′ chapter 16). Phosphate group H

C

C

P

5′

H3C

N

C

C

N

H

H

C

C

N

H

O 1′

4′ 3′

Phosphodiester bonds

2′

P

5′

O

4′

Sugar–phosphate “backbone”

1′ 3′

P

2′

O P

5′

O 1′

4′

5-carbon sugar

A

O

T

3′

2′

P

O

Nitrogenous base

5′

O

1′

4′ 3′

OH

2′

C Phosphodiester bonds

P

T

O

O P

5′ end

A P

Hydrogen bonds between nitrogenous bases

O

3′

OH

Figure 3.16 The structure of a polynucleotide chain. 

In a nucleic acid, nucleotides are linked to one another via phosphodiester bonds formed between the phosphate of one nucleotide and the sugar of the next nucleotide. We call this the sugar-phosphate backbone, with the organic bases protruding from this chain. The backbone also has different ends: a 5´ phosphate end and a 3´ hydroxyl end (the blue numbers come from the numbers in the sugars).

P

G

3′ end

Figure 3.17 The structure of DNA. DNA consists of two polynucleotide chains running in opposite directions wrapped about a single helical axis. Hydrogen bond formation (dashed lines) between the nitrogenous bases, called base-pairing, causes the two chains of DNA to bind to each other and form a double helix. chapter

3 The Chemical Building Blocks of Life 45

Nitrogenous base (adenine)

P P

P

T

T

G

P

A

Deoxyribosephosphate backbone

P

C

P

O P

P

G

Hydrogen bonding occurs between base-pairs P

P

P

7

P

C

O−

P

N1

8

O O

6

5

O

5′

CH2

O−

triphosphate (ATP) contains adenine, a 5-carbon sugar, and three phosphate groups.

9

N

4

N

2 3

O 4′

1′ 3′

2′

OH OH 5-carbon sugar

A nucleic acid is a polymer composed of alternating phosphate and 5-carbon sugar groups with a nitrogenous base protruding from each sugar. In DNA, this sugar is deoxyribose. In RNA, the sugar is ribose. RNA also contains the base uracil instead of thymine. DNA is a double-stranded helix that stores hereditary information as a specific sequence of nucleotide bases. RNA has multiple roles in a cell, including carrying information from DNA and forming part of the ribosome.

Ribose-phosphate backbone

G P

U A Bases

U

P

■■

P RNA

P

N

Learning Outcomes Review 3.3

P

A

O O

Figure 3.19 ATP. Adenosine

A

T

P

P

P O−

A

C Bases

O

P

P

P

P

Triphosphate group

G A

DNA

NH2

If an RNA molecule is copied from a DNA strand, what is the relationship between the sequence of bases in RNA and each DNA strand?

G

Figure 3.18 DNA versus RNA. DNA forms a double helix, uses deoxyribose as the sugar in its sugar–phosphate backbone, and uses thymine among its nitrogenous bases. RNA is usually singlestranded, uses ribose as the sugar in its sugar–phosphate backbone, and uses uracil in place of thymine.

3.4  Proteins:

Molecules with Diverse Structures and Functions

Learning Outcomes

Other nucleotides are vital components of energy reactions Nucleotides play other critical roles in cells. For example, adenosine triphosphate (ATP; figure 3.19) is called the energy currency of the cell. Cells use the energy released by breaking down food molecules to synthesize ATP (discussed in detail in chapter 7). Cells can then use the energy released by ATP hydrolysis in a variety of transactions, the way we use money in society. Energy from ATP hydrolysis is used to drive energetically unfavorable chemical reactions, to power transport across membranes, and to power the movement of cells. Two other important nucleotide-containing molecules are nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD). These molecules function as electron carriers in a variety of cellular processes. You will see the action of these molecules in detail when we discuss photosynthesis and respiration (see chapters 7 and 8). 46 part I The Molecular Basis of Life

1. Describe the possible levels of protein structure. 2. Explain how motifs and domains contribute to protein structure. 3. Understand the relationship between amino acid sequence and their three-dimensional structure.

Proteins are the most diverse group of biological macromolecules, both chemically and functionally. Because proteins have so many different functions in cells we could not begin to list them all. We can, however, group these functions into the following seven categories. This list is a summary only, however; the function of proteins is relevant to most topics in biology. 1. Enzyme catalysis. Enzymes are biological catalysts that facilitate specific chemical reactions. Because of this property, the appearance of enzymes was one of the most important events in the evolution of life. Most enzymes are three-dimensional globular proteins that fit snugly

around the molecules they act on. This fit facilitates chemical reactions by stressing particular chemical bonds. 2. Defense. Other globular proteins use their shapes to “recognize” foreign microbes and cancer cells. These cell-surface receptors form the core of the body’s ­endocrine and immune systems. 3. Transport. A variety of globular proteins transport small molecules and ions. The transport protein ­hemoglobin, for example, transports oxygen in the blood. Membrane transport proteins help move ions and molecules across the membrane. 4. Support. Protein fibers play structural roles. These fibers include keratin in hair, fibrin in blood clots, and collagen. The last one, collagen, forms the matrix of skin, ligaments, tendons, and bones and is the most abundant protein in a vertebrate body. TA B L E 3 . 2

Defense

Transport

Class of Protein Enzymes

Motion Regulation

Storage

Examples

Examples of Use

Glycosidases

Cleave polysaccharides

Proteases

Break down proteins

Polymerases

Synthesize nucleic acids

Kinases

Phosphorylate sugars and proteins

Immunoglobulins

Antibodies

Mark foreign proteins for elimination

Toxins

Snake venom

Blocks nerve function

Cell-surface antigens

MHC* proteins

“Self”-recognition

Circulating transporters

Hemoglobin

Carries O2 and CO2 in blood

Myoglobin

Carries O2 and CO2 in muscle

Cytochromes

Electron transport

Sodium–potassium pump

Excitable membranes

Proton pump

Chemiosmosis

Glucose transporter

Transports glucose into cells

Collagen

Forms cartilage

Keratin

Forms hair, nails

Fibrin

Forms blood clots

Actin

Contraction of muscle fibers

Myosin

Contraction of muscle fibers

Osmotic proteins

Serum albumin

Maintains osmotic concentration of blood

Gene regulators

lac Repressor

Regulates transcription

Hormones

Insulin

Controls blood glucose levels

Vasopressin

Increases water retention by kidneys

Oxytocin

Regulates uterine contractions and milk production

Ferritin

Stores iron, especially in spleen

Casein

Stores ions in milk

Calmodulin

Binds calcium ions

Membrane transporters

Support

Table 3.2 summarizes these functions and includes examples of the proteins that carry them out in the human body.

The Many Functions of Protein

Function Enzyme catalysis

5. Motion. Muscles contract through the sliding motion of two kinds of protein filaments: actin and myosin. Contractile proteins also play key roles in the cell’s cytoskeleton and in moving materials within cells. 6. Regulation. Small proteins called hormones serve as intercellular messengers in animals. Proteins also play many regulatory roles within the cell—turning on and shutting off genes during development, for example. In addition, proteins receive information, acting as cell-surface receptors. 7. Storage. Calcium and iron are stored in the body by binding as ions to storage proteins.

Fibers

Muscle

Ion-binding

*MHC, major histocompatibility complex.

chapter

3 The Chemical Building Blocks of Life 47

Proteins are polymers of amino acids Proteins are linear polymers made with 20 different amino acids. Amino acids, as their name suggests, contain an amino group (—NH2) and an acidic carboxyl group (—COOH). The specific order of amino acids determines the protein’s structure and function. Many scientists believe amino acids were among the first molecules formed on the early Earth. It seems highly likely that the oceans that existed early in the history of the Earth contained a wide variety of amino acids.

Amino acid structure The generalized structure of an amino acid is shown as amino and carboxyl groups bonded to a central carbon atom, with an additional hydrogen and a functional side group i­ndicated by R. These components completely fill the bonds of the central carbon: R H2N— C— COOH H The unique character of each amino acid is determined by the nature of the R group. Notice that unless the R group is an H atom, as in glycine, amino acids are chiral and can exist as two enantiomeric forms: d or l. In living systems, only the l-amino acids are found in proteins, and d-amino acids are rare. The R group also determines the chemistry of amino acids. Serine, in which the R group is —CH2OH, is a polar molecule. Alanine, which has —CH3 as its R group, is nonpolar. The 20 common amino acids are grouped into five chemical classes, based on their R group (see figure 3.21): 1. Nonpolar amino acids, such as leucine, often have R groups that contain —CH2 or —CH3. 2. Polar uncharged amino acids, such as threonine, have R groups that contain oxygen (or —OH). 3. Charged amino acids, such as glutamic acid, have R groups that contain acids or bases that can ionize. 4. Aromatic amino acids, such as phenylalanine, have R groups that contain an organic (carbon) ring with alternating single and double bonds. These are also nonpolar. 5. Amino acids that have special functions have unique properties. Some examples are methionine, which is often the first amino acid in a chain of amino acids; proline, which causes kinks in chains; and cysteine, which links chains together. Each amino acid affects the shape of a protein differently, depending on the chemical nature of its side group. For example, portions of a protein chain with numerous nonpolar amino acids tend to fold into the interior of the protein by hydrophobic exclusion.

H

R

H +N

C

C

H

H

O

O–

H

H

R

N

C

C

H

H

O

+

Amino acid

O–

Amino acid H2O H

R

H +N

C

C

H

H

O

H

R

N

C

C

H

O

O–

Dipeptide

Figure 3.20 The peptide bond. A peptide bond forms when the amino end of one amino acid joins to the carboxyl end of another. Reacting amino and carboxyl groups are shown in red and nonreacting groups are highlighted in green. Notice that the resulting dipeptide still has an amino end and a carboxyl end. Because of the partial double-bond nature of peptide bonds, the resulting peptide chain cannot rotate freely around these bonds.

groups on a pair of amino acids can undergo a dehydration reaction to form a covalent bond. The covalent bond that links two amino acids is called a peptide bond (figure 3.20). The two amino acids linked by such a bond are not free to rotate around the N—C linkage because the peptide bond has a partial ­ double-bond ­character. This is different from the N—C and C—C bonds to the central carbon of the amino acid. This lack of rotation about the peptide bond is one factor that determines the structural character of the coils and other regular shapes formed by chains of amino acids. An unbranched chain of amino acids linked by peptide bonds is called a polypeptide. If a polypeptide can fold into a structure with some function, such as the antimicrobial enzyme lysozyme in your saliva, we also call it a protein. However, some proteins, such as the oxygen-carrying protein hemoglobin in your blood, consist of more than one polypeptide. The pioneering work of Frederick Sanger in the early 1950s provided the evidence that each kind of protein has a specific amino acid sequence. Using chemical methods to remove successive amino acids and then identify them, Sanger succeeded in determining the amino acid sequence of insulin. In so doing he demonstrated clearly that this protein had a defined sequence, which was the same for all insulin molecules in the solution. Although many different amino acids occur in nature, only 20 commonly occur in proteins. Of these 20, 8 are called essential amino acids because humans cannot synthesize them and thus must get them from their diets. Figure 3.21 illustrates these 20 amino acids and their side groups.

Peptide bonds

Proteins have levels of structure

In addition to its R group, each amino acid is ionized at physiological pH, with a positive amino (NH3+) group at one end and a negative carboxyl­(COO−) group at the other. The amino and carboxyl

The shape of a protein determines its function. One way to study the shape of something as small as a protein is to look at it with very short wavelength energy—in other words, with X-rays. X-rays

48 part I The Molecular Basis of Life

Nonpolar

Polar uncharged

CH3 H3N+

OH

C

C

H

O

O– H3N+ CH3

CH3

H3N+

C

C

H

O H3N+ CH3

O–

C

H O Valine (Val)

CH3

Nonaromatic

O–

C

Serine (Ser)

CH

H

C

OH

H3N+

C

C

C

CH3

H3N+

C

C

O O–

H O Isoleucine (Ile)

CH3

CH3

H3N+

CH CH2 H3N+

H C

C

H

O

C

O–

C

NH2

NH2

CH2

C

CH2

NH

C

C

H

O

CH2

CH2 O–

C

H

O

O

H3N+

NH2 C

Asparagine (Asn)

C

C

H

O

CH2 CH2

O–

O–

NH3+

CH2 CH2 CH2

C

C

H

O

H3N+

O–

C

C

H

O

O–

Lysine (Lys)

Glutamine (Gln)

Glycine (Gly)

C

CH2

CH2 H3N+

C

H O Arginine (Arg)

Aspartic acid (Asp)

CH2

NH2+

CH2

O–

O C

C

O–

H3N+

C

H O Leucine (Leu)

O–

O–

C

Glutamic acid (Glu)

H O Threonine (Thr)

CH2 H

O–

O

CH2

Alanine (Ala)

H3N+

Charged

OH HC

Aromatic

CH2 H3N+

C

C

H

O

C

C

O–

C

C

H

O

O–

H3N+

C

Special function

CH2

CH

C

+

NH2

O Proline (Pro)

O–

H3N+

CH

O–

H3

N+

C

C

H

O

O–

Histidine (His)

Figure 3.21 The 20 common amino acids. Each

CH3

CH2

C

H O Tyrosine (Tyr)

Tryptophan (Trp)

N H

CH2

CH2

CH2 H3N+

Phenylalanine (Phe)

CH2

NH+

NH

S

H

CH2

S

CH2

CH2

C

C

H

O

Methionine (Met)

O–

NH3+

C

C

H

O

Cysteine (Cys)

O–

amino acid has the same chemical backbone, but differs in the side, or R, group. Seven of the amino acids are nonpolar because they have —CH 2 or —CH 3 in their R groups. Two of the seven contain ring structures with alternating double and single bonds, which classifies them also as aromatic. Another five are polar because they have oxygen or a hydroxyl group in their R groups. Five others are capable of ionizing to a charged form. The remaining three special-function amino acids have chemical properties that allow them to help form links between protein chains or kinks in proteins. chapter

3 The Chemical Building Blocks of Life 49

can be passed through a crystal of protein to produce a diffraction pattern. This pattern can then be analyzed by a painstaking procedure that allows the investigator to build up a three-dimensional picture of the position of each atom. The first protein to be analyzed in this way was myoglobin, and the related protein hemoglobin was analyzed soon thereafter. As more and more proteins were studied, a general principle became evident: in every protein studied, essentially all the internal amino acids are nonpolar ones—amino acids such as leucine, valine, and phenylalanine. Water’s tendency to hydrophobically exclude nonpolar molecules literally shoves the nonpolar portions of the amino acid chain into the protein’s interior (see chapter 2). This tendency forces the nonpolar amino acids into close contact with one another, leaving little empty space inside. Polar and charged amino acids are restricted to the surface of the protein, except for the few that play key functional roles.

Primary Structure

R C

C

H

O

H

H

O

N

C

C

R

R N

C

C

H

H

O

H

H

N

C R

The primary structure can fold into a pleated sheet, or turn into a helix

Secondary Structure

The structure of proteins is usually discussed in terms of a hierarchy of four levels: primary, secondary, tertiary, and quaternary (figure 3.22). We will examine this view and then integrate it with a more modern approach arising from our increasing knowledge of protein structure.

Primary structure: Amino acid sequence The primary structure of a protein is its amino acid sequence. Because the R groups that distinguish the amino acids play no role in the peptide backbone of proteins, a protein can consist of any sequence of amino acids. Thus, because any of 20 different amino acids might appear at any position, a protein containing 100 amino acids could form any of 20100 different amino acid sequences (that’s the same as 10130, or 1 followed by 130 zeros—more than the number of atoms known in the universe). This important property of proteins permits great diversity.

Figure 3.22 Levels of protein structure. The primary structure of a protein is its amino acid sequence. Secondary structure results from hydrogen bonds forming between nearby amino acids. This produces two different kinds of structures: beta (β) pleated sheets, and coils called alpha (α) helices. The tertiary structure is the final 3-D shape of the protein. This determines how regions of secondary structure are then further folded in space to form the final shape of the protein. Quaternary structure is found only in proteins with multiple polypeptides. In this case the final structure of the protein is the arrangement of the multiple polypeptides in space. Secondary Structure

• • •

• • •

• • •

• • •

• • • • • •

• • •

• • •

• • • • • •

• • •

• • •

• • •

β-pleated sheet

Tertiary Structure

50 part I The Molecular Basis of Life

α-helix

Quaternary Structure

Consider the protein hemoglobin, the protein your blood uses to transport oxygen. Hemoglobin is composed of two α-globin peptide chains and two β-globin peptide chains. The α-globin chains differ from the β-globin ones in the sequence of amino acids. Furthermore, any alteration in the normal sequence of either of the types of globin proteins, even by a single amino acid, can have drastic effects on how the protein functions.

sections of peptide are oriented in the same direction or the opposite direction. These two kinds of secondary structure create regions of the protein that are cylindrical (α helices) and planar (β sheets). A protein’s final structure can include regions of each type of secondary structure. For example, DNA-binding proteins usually have regions of α helix that can lie across DNA and interact directly with the bases of DNA. Porin proteins that form holes in membranes are composed of β sheets arranged to form a pore in the membrane. Finally, in hemoglobin, the α- and β-globin peptide chains that make up the final molecule each have characteristic regions of secondary structure.

Secondary structure: Hydrogen bonding patterns The amino acid side groups are not the only portions of proteins that form hydrogen bonds. The polypeptide backbone consists of repeating units of amino acids, each with an amino and a carboxyl group that can participate in hydrogen bonds. These hydrogen bonds can be with water or with other amino acids in the backbone. If too many hydrogen bonds formed with water, the protein would tend to behave like a random coil and wouldn’t produce the kinds of globular structures that are common in proteins. Linus Pauling suggested that the amino and carboxyl groups could interact with one another if the peptide was coiled into a spiral that he called the α helix. We now call this sort of regular interaction of groups in the peptide backbone secondary structure. Another form of secondary structure can occur between regions of peptide aligned next to each other to form a planar structure called a β sheet. These can be either parallel or antiparallel depending on whether the adjacent

Tertiary structure: Folds and links The final folded shape of a globular protein is called its tertiary structure. This tertiary structure contains regions that have secondary structure and determines how these are further arranged in space to produce the overall structure. A protein is initially driven into its tertiary structure by hydrophobic exclusion from water. Ionic bonds between oppositely charged R groups bring regions into close proximity, and disulfide bonds (covalent links between two cysteine R groups) lock particular regions together. The final folding of a protein is determined by its primary structure—the chemical nature of its side groups (see figures 3.22 and 3.23).

O

C C

H

C

O

C

N

O

C

N

(CH2)4 O

N

H C

H S

H

S

C

N

Ionic bond

C

H

C

O

O

C

R

R

C

C

H

R

N

C

C

Disulfide bridge

NH3+

O

O−

C

C

N

CH2

Hydrogen bond

N

C

N

C CH3

CH3

van der Waals attraction

C C

O

C

O

a.

b.

CH3

CH3

H

Hydrophobic exclusion

CH3 CH3

C CH3

CH3

e.

c.

CH2 C

CH3

d.

Figure 3.23 Interactions that contribute to a protein’s shape. Aside from the bonds that link together the amino acids in a protein, several other weaker forces and interactions stabilize protein structure. a. Hydrogen bonds can form between amino and carboxyl groups of the peptide backbone and between R groups of amino acids. b. Covalent disulfide bridges can form between two cysteine side chains. c. Ionic bonds can form between groups with opposite charge. d. van der Waals attractions, which are weak attractions between atoms due to oppositely polarized electron clouds, can occur. e. Polar portions of the protein tend to gather on the outside of the protein and interact with water, whereas the hydrophobic portions of the protein, including nonpolar amino acid chains, are shoved toward the interior of the protein. chapter

3 The Chemical Building Blocks of Life 51

Many small proteins can be fully unfolded (“denatured”) and will spontaneously refold into their characteristic shape. Other larger proteins tend to associate together and form insoluble clumps when denatured, such as the film that can form when you heat milk for hot chocolate. The tertiary structure is stabilized by a number of forces including hydrogen bonding between R groups of different amino acids, electrostatic attraction between R groups with opposite charge (also called salt bridges), hydrophobic exclusion of nonpolar R groups, and covalent bonds in the form of disulfides. The stability of a protein, once it has folded into its tertiary shape, is strongly influenced by how well its interior fits together. When two nonpolar chains in the interior are very close together, they experience a form of molecular attraction called van der Waals forces. Individually quite weak, these forces can add up to a strong attraction when many of them come into play, like the combined strength of hundreds of hooks and loops on a strip of Velcro. These forces are effective only over short distances, however. No “holes” or cavities exist in the interior of proteins. The variety of different nonpolar amino acids, with a different-sized R group with its own distinctive shape, allows nonpolar chains to fit very precisely ­within the protein interior. It is therefore not surprising that changing a single amino acid can drastically alter the structure, and thus the function, of a protein. The sickle cell version of hemoglobin (HbS), for example, is a change of a single glutamic acid for a valine in the β-globin chain (see chapter 15). This change substitutes a charged amino acid for a nonpolar one on the surface of the protein, leading the protein to become sticky and form clumps. Another variant of hemoglobin called HbE, actually the most common in human populations, causes a change from glutamic acid to lysine at a different site in the β-globin chain. In this case the structural change is not as dramatic, but it still impairs function, resulting in blood disorders called anemia and thalassemia. More than 700 structural variants of hemoglobin are known, with up to 7% of the world’s population being carriers of forms that are medically important.

Quaternary structure: Subunit arrangements When two or more polypeptide chains associate to form a functional protein, the individual chains are referred to as subunits of the protein. The arrangement of these subunits is termed its quaternary structure. In proteins composed of subunits, the interfaces where the subunits touch one another are often nonpolar, and they play a key role in transmitting information between the subunits about individual subunit activities. Remember that the protein hemoglobin is composed of two α-chain subunits and two β-chain subunits. Each α- and β-globin chain has a primary structure consisting of a specific sequence of amino acids. This then assumes a characteristic secondary structure consisting of α helices and β sheets that are then arranged into a specific tertiary structure for each α- and β-globin subunit. Lastly, these subunits are then arranged into their final quaternary structure. This is the final structure of the protein. For proteins that consist of only a single peptide chain, the enzyme lysozyme for example, the tertiary structure is the final structure of the protein. 52 part I The Molecular Basis of Life

Motifs and domains are structural elements of proteins To directly determine the sequence of amino acids in a protein is a laborious task. Although the process has been automated, it remains slow and difficult. The ability to sequence DNA changed this situation rather suddenly. Originally done manually, the Human Genome Project drove the development of automated sequencing. This increased throughput significantly, but the advent of next-generation sequencing technologies resulted in quantum increases in sequence data. Today, over 40,000 bacterial genomes have been sequenced, and almost 8000 eukaryotic genomes, including more than 80 mammalian genomes. Because the DNA sequence is directly related to amino acid sequence in proteins, biologists now have an enormous database of protein sequences to compare and analyze. This new information has also stimulated thought about the logic of the genetic code and whether underlying patterns exist in protein structure. Our view of protein structure has evolved with these data. Researchers still view the four-part hierarchical structure as important, but two additional terms have entered the biologist’s vocabulary: motif and domain.

Motifs As biologists discovered the three-dimensional structure of proteins (an even more laborious task than determining the sequence), they noticed similarities between otherwise dissimilar proteins. These similar structures are called motifs, or sometimes “supersecondary structure.” The term motif is borrowed from the arts and refers to a recurring thematic element in music or design. One very common protein motif is the β-α-β motif, which creates a fold or crease: the so-called “Rossmann fold” at the core of nucleotide-binding sites in a wide variety of proteins. A second motif that occurs in many proteins is the β barrel, which is a β sheet folded around to form a tube. A third type of motif, the helix-turn-helix, consists of two α helices separated by a bend. This motif is important because many proteins use it to bind to the DNA double helix (figure 3.24; see also chapter 16). Motifs indicate a logic to structure that investigators still do not understand. Do they simply represent a reuse by evolution of something that already works, or are they an optimal solution to a problem, such as how to bind a nucleotide? One way to think about it is that if amino acids are letters in the language of proteins, then motifs represent repeated words or phrases. Motifs have been useful in determining the function of unknown proteins. The sequence of an unknown protein can be compared to a database of known functional motifs. Finding a characterized motif in an unknown protein can shed light on that protein’s function.

Domains Domains of proteins are functional units within a larger structure. They can be thought of as substructure within the tertiary structure of a protein (figure 3.24). To continue the metaphor: amino acids are letters in the protein language, motifs are words or phrases, and domains are paragraphs.

Motifs

Domains

Domain 1

β-α-β motif

helix-turn-helix motif

Figure 3.24 Motifs and domains. The elements of

secondary structure can combine, fold, or crease to form motifs. These motifs are found in different proteins and can be used to predict function. Proteins also are made of larger domains, which are functionally distinct parts of a protein. The arrangement of these domains in space is the tertiary structure of a protein. Domain 3

Most proteins are made up of multiple domains that perform different parts of the protein’s function. In many ­cases, these domains can be physically separated. For example, transcription factors (discussed in chapter 16) are proteins that bind to DNA and initiate its transcription. If the DNA-binding region is exchanged with a different transcription factor, then the specificity of the factor for DNA can be changed without changing its ability to stimulate transcription. Such “domain-swapping” experiments have been performed with many transcription factors, and they indicate, among other things, that the DNA-binding and activation domains are functionally separate. These functional domains of proteins may also help the protein to fold into its proper shape. As a polypeptide chain folds, the domains take their proper shape, each more or less independently of the others. This action can be demonstrated­experimentally by artificially producing the fragment of a polypeptide that forms the domain in the intact protein, and showing that the fragment folds to form the same structure it exhibits in the intact protein. A single polypeptide chain connects the domains of a protein, like a rope tied into several adjacent knots. Domains can also correspond to the structure of the genes that encode them. Later, in chapter 15, you will see that genes in eukaryotes are often in pieces within the genome, and these pieces, called exons, sometimes encode the functional domains of a protein. This finding led to the idea that evolution can act by shuffling protein-encoding domains.

The process of folding relies on chaperone proteins Originally, biochemists thought that newly made proteins fold spontaneously, randomly trying out different configurations as

Domain 2

hydrophobic interactions with water shove nonpolar amino acids into the protein’s interior until the final structure is arrived at. We now know this view is too simple. Protein chains can fold in so many different ways that trial and error would simply take too long. In addition, as the open chain folds its way toward its final form, nonpolar “sticky” interior portions are exposed during intermediate stages. If these intermediate forms are placed in a test tube in an environment identical to that inside a cell, they stick to other, unwanted protein partners, forming a gluey mess. How do cells avoid having their proteins clump into a mass? An early clue came with the discovery of mutations in bacterial cells that prevent the assembly of replicating viruses. The mutations affected a newly identified class of proteins named chaperone proteins, for their ability to accompany a protein on its path to a properly folded state. Molecular biologists have now identified many proteins that act as molecular chaperones. This large class of proteins can be divided into subclasses, and representatives have been found in essentially every organism that has been examined. At least some of these proteins have been shown to be necessary for viability, illustrating their fundamental importance. Many are so-called heat shock proteins, produced in large amounts in response to elevated temperature. High temperatures cause proteins to unfold, and heat shock chaperone proteins help the cell’s proteins to refold properly. One class of these proteins, called chaperonins, has been extensively studied. In the bacterium Escherichia coli (E. coli), one example is the essential protein GroE chaperonin. In mutants in which the GroE chaperonin is inactivated, fully 30% of the bacterial proteins fail to fold properly. Chaperonins associate to form a large macromolecular complex that resembles a cylindrical chapter

3 The Chemical Building Blocks of Life 53

Misfolded protein

Chaperone protein

Cap

ATP ADP + P

Correctly folded protein

Chance for protein to refold

Figure 3.25 How one type of chaperone protein works. This barrel-shaped chaperonin is from the GroE family of chaperone

proteins. It is composed of two identical rings each with seven identical subunits, each of which has three distinct domains. An incorrectly folded protein enters one chamber of the barrel, and a cap seals the chamber. Energy from the hydrolysis of ATP fuels structural alterations to the chamber, changing it from hydrophobic to hydrophilic. This change allows the protein to refold. After a short time, the protein is ejected, either folded or unfolded, and the cycle can repeat itself.

container. Proteins can move into the container, and the container itself can change its shape considerably (­figure 3.25). Experiments have shown that an improperly folded protein can enter the chaperonin and be refolded. Although we don’t know exactly how this happens, it seems to involve changes in the hydrophobicity of the interior of the chamber. The flexibility of the structure of chaperonins is amazing. We tend to think of proteins as being fixed structures, but this is clearly not the case for chaperonins and this flexibility is necessary for their function. It also illustrates that even domains that may be very widely separated in a very large protein are still functionally connected. The folding process within a chaperonin harnesses the hydrolysis of ATP to power these changes in structure necessary for function. This entire process can occur in a cyclic manner until the appropriate structure is achieved. Cells use these chaperonins both to accomplish the original folding of some proteins and to restore the structure of incorrectly folded ones.

Improper folding of proteins can result in disease Chaperone protein deficiencies may be implicated in certain diseases in which key proteins are improperly folded. Cystic fibrosis is a hereditary disorder in which a mutation disables a vital protein that moves ions across cell membranes. As a result, people with cystic fibrosis have thicker than normal mucus. This results in breathing problems, lung disease, and digestive difficulties, among other things. One interesting feature of the molecular analysis of this disease has been the number of different mutations found in human populations. Many of these mutations affect the ability of the encoded protein to fold properly. The number of different mutations that can result in improperly folded proteins may be related to the fact that the native protein often fails to fold properly. 54 part I The Molecular Basis of Life

Denaturation inactivates proteins If a protein’s environment is altered, the protein may change its shape or even unfold completely. This process is called ­denaturation (figure 3.26). Proteins can be denatured when the pH, temperature, or ionic concentration of the surrounding solution changes.

Properly folded protein

Denaturation

Denatured protein

Figure 3.26 Protein denaturation. Environmental changes, such as variation in temperature or pH, can cause a protein to unfold and lose its shape. This loss of structure is called denaturation. Denatured proteins are biologically inactive.

Denatured proteins are usually biologically inactive. This action is particularly significant in the case of enzymes. Because practically every chemical reaction­in a living organism is catalyzed by a specific ­enzyme, it is vital that a cell’s enzymes work properly. The traditional methods of food preservation, salt curing, and pickling involve denaturation of proteins. Prior to the general availability of refrigerators and freezers, the only practical way to keep microorganisms from growing in food was to keep the food in a solution containing a high concentration of salt or vinegar, which denatured the enzymes of most microorganisms and prevented them from growing on the food. Most enzymes function within a very narrow range of environmental conditions. Blood-borne enzymes that course through a human body at a pH of about 7.4 would rapidly become denatured in the highly acidic environment of the stomach. Conversely, the protein-degrading enzymes that function at a pH of 2 or less in the stomach would be denatured in the relatively basic pH of the blood. Similarly, organisms that live near oceanic hydrothermal vents have enzymes that work well at these extremes of temperature (over 100°C). They cannot survive in cooler waters, because their enzymes do not function properly at lower temperatures. Any ­given organism usually has a tolerance range of pH, temperature, and salt concentration. Within that range, its enzymes maintain the proper shape to carry out their biological functions.

When a protein’s normal environment is reestablished after denaturation, a small protein may spontaneously refold into its natural shape, driven by the interactions between its nonpolar amino acids and water (figure 3.27). This process is termed renaturation, and it was first established for the enzyme ribonuclease (RNase). The renaturation of RNase led to the doctrine that primary structure determines tertiary structure. Larger proteins can rarely refold spontaneously, however, because of the complex nature of their final shape, so this simple idea needs to be qualified. The fact that some proteins can spontaneously renature implies that tertiary structure is strongly influenced by primary structure. In an extreme example, the E. coli ribosome can be taken apart and put back together experimentally. Although this process requires temperature and ion concentration shifts, it indicates an amazing degree of self-assembly. That complex structures can arise by self-assembly is a key idea in the study of modern biology. It is important to distinguish denaturation from dissociation. For proteins with quaternary structure, the subunits may be dissociated (separated) without losing their individual tertiary structure. For example, the four subunits of hemoglobin may dissociate into four individual molecules (two α-globins and two β-globins) without denaturation of the folded globin proteins. They readily reassume their four-subunit quaternary structure.

SCIENTIFIC THINKING Hypothesis: The 3-D structure of a protein is the thermodynamically stable structure. It depends only on the primary structure of the protein and the solution conditions. Prediction: If a protein is denatured and allowed to renature under native conditions, it will refold into the native structure. Test: Ribonuclease is treated with a reducing agent to break disulfide bonds and is then treated with urea to completely unfold the protein. The disulfide bonds are re-formed under nondenaturing conditions to see if the protein refolds properly. Native ribonuclease

Unfolded ribonuclease

Reduced ribonuclease

Reducing agents

Heating or addition of urea

Oxidizing agents

Cooling or removal of urea

S S disulfide bonds

Broken disulfide bonds (SH)

Result: Denatured ribonuclease refolds properly under nondenaturing conditions. Conclusion: The hypothesis is supported. The information in the primary structure (amino acid sequence) is sufficient for refolding to occur. This implies that protein folding results in the thermodynamically stable structure. Further Experiments: If the disulfide bonds were allowed to re-form under denaturing conditions, would we get the same result? How can we rule out that the protein had not been completely denatured and therefore retained some structure?

Figure 3.27 Primary structure determines tertiary structure. chapter

3 The Chemical Building Blocks of Life 55

Learning Outcomes Review 3.4

Proteins are made of 20 different amino acids, and have diverse functions. Protein structure is a hierarchy with: (1) primary structure, the sequence of amino acids; (2) secondary structure, regular coils and sheets; (3) tertiary structure, the actual threedimensional shape; and (4) quaternary structure, the association of multiple polypeptide subunits. Different proteins have similar substructures called motifs, and can be broken down into functional domains. Proteins function within a narrow range of conditions; outside this they will unfold or denature and lose function. Changing conditions can allow for renaturation; this implies that amino acid sequence determines structure. ■■

How does our knowledge of protein structure help us to predict the function of unknown proteins?

3.5  Lipids:

Hydrophobic Molecules

Learning Outcomes 1. Describe the structure of triglycerides. 2. Explain how fats function as energy-storage molecules. 3. Apply knowledge of the structure of phospholipids to the formation of membranes.

Lipids are a somewhat loosely defined group of molecules with one main chemical characteristic: they are insoluble in water. Storage fats such as animal fat are one kind of lipid. Oils such as those from olives, corn, and coconut are also lipids, as are waxes such as beeswax and earwax. Even some vitamins are lipids! Lipids have a very high proportion of nonpolar carbon– hydrogen (C—H) bonds, and so long-chain lipids cannot fold up like a protein to confine their nonpolar portions away from the surrounding aqueous environment. Instead, when they are placed in water, many lipid molecules spontaneously cluster together and expose what polar (hydrophilic) groups they have to the surrounding water, while confining the nonpolar (hydrophobic) parts of the molecules together within the cluster. You may have noticed this effect when you add oil to a pan containing water, and the oil beads up into cohesive drops on the water’s surface. This spontaneous assembly of lipids is of paramount importance to cells, as it underlies the structure of cellular membranes.

Fats consist of fatty acid polymers attached to glycerol Many lipids are built from a simple skeleton made up of two main kinds of molecules: fatty acids and glycerol. Fatty acids are long-chain hydrocarbons with a carboxyl group (COOH) at one 56 part I The Molecular Basis of Life

end. Glycerol is a 3-carbon polyalcohol (three —OH groups). Many lipid molecules consist of a glycerol molecule with three fatty acids attached, one to each carbon of the glycerol backbone. Because it contains three fatty acids, a fat molecule is commonly called a triglyceride (the more accurate chemical name is triacylglycerol). This basic structure is depicted in f­ igure 3.28. The three fatty acids of a triglyceride need not be identical, and often they are very different from one another. The hydrocarbon chains of fatty acids vary in length. The most common are even-numbered chains of 14 to 20 carbons. The many C—H bonds of fats serve as a form of long-term energy storage. If all of the internal carbon atoms in a fatty acid chain are bonded to two hydrogen atoms, we call this saturated, with the maximum number of hydrogen atoms possible (see figure 3.28). A fatty acid with double bonds between one or more pairs of successive carbon atoms will have fewer hydrogen atoms, and is thus unsaturated. Fatty acids with one double bond are called monounsaturated, and those with more than one double bond are termed polyunsaturated. Most naturally occurring unsaturated fatty acids have double bonds with a cis configuration, where the carbon chain is on the same side before and after the double bond (in figure 3.28b all double bonds are cis). If the carbon chain is on the opposite side before and after the double bond, it is a trans configuration. The presence of a double bond in fatty acids affects the melting point of a triglyceride because the lack of free rotation about cis C=C double bonds produces a “kink” in the chain. This makes unsaturated fats liquid at room temperature (oils), while most saturated fats, such as animal fat and those in butter, are solid at room temperature. For many years the consumption of saturated animal fats was linked to increased risk of cardiovascular disease. Because of this, the food industry started producing solid products, such as margarine, by adding hydrogen to unsaturated fats to make them partially saturated. This process, called partial hydrogenation, was intended to produce a healthier product. Unfortunately, the process also converts cis double bonds to trans double bonds, creating so-called “trans fats.” Because they are less “kinked,” they are solid at room temperature, but also have been linked to elevated levels of lowdensity lipoprotein (LDL) “bad cholesterol,” and lowered levels of high-density lipoprotein (HDL) “good cholesterol.” So the risk for cardiovascular disease from trans fats appears to be greater than that due to saturated fat. The attempt to produce something more healthy actually produced something less healthy. Trans fats have been removed from the FDA’s list of “generally regarded as safe” (GRAS) compounds, which has helped to remove trans fats from most common sources. Placed in water, triglycerides spontaneously associate together, forming fat globules that can be very large relative to the size of the individual molecules. Because fats are insoluble in water, they can be deposited at specific locations within an organism, such as in vesicles of adipose tissue. Organisms contain many other kinds of lipids besides fats (figure 3.29). Terpenes are long-chain lipids that are components of many biologically important pigments, such as chlorophyll and the visual pigment retinal. Rubber is also a terpene. Steroids, another class of lipid, are composed of four carbon rings. Most animal cell membranes contain the steroid cholesterol. Other

Structural Formula H

Structural Formula H

O H H H H H H H H H H H H H H H H H

H C O C C C C C C C C C C C C C C C C C C H

O H H H H H H H

H H H H H H H H

H C O C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H H H H H H H

H H H H H H H H H H H H H H H H H

O H H H H H H H

O H H H H H H H H H H H H H H H H H H

H H H H H

H H H H H H H H H H H H H H H H H

O H H H H H H H H H H H H H H H H H

H C O C C C C C C C C C C C C C C C C C C H

H

H C O C C C C C C C C C C C C C C C C C C H

H H H H H H H H H H H H H H H H H

H C O C C C C C C C C C C C C C C C C C C H

O H H H H H H H

H

H

H H

H C O C C C C C C C C C C C C C C C C C C H H

H H H H H H H H H H H H H H H H H

Space-Filling Model

H H H H H H H H H H H H H H H H H

Space-Filling Model

b.

a.

Figure 3.28 Saturated and unsaturated fats. a. A saturated fat is composed of triglycerides that contain three saturated fatty acids

(the kind that have no double bonds). A saturated fat therefore has the maximum number of hydrogen atoms bonded to its carbon chain. Most animal fats are saturated. b. Unsaturated fat is composed of triglycerides that contain three unsaturated fatty acids (the kind that have one or more double bonds). These have fewer than the maximum number of hydrogen atoms bonded to the carbon chain. This example includes both a monounsaturated and two polyunsaturated fatty acids. Plant fats are typically unsaturated. The many kinks of the double bonds prevent the triglyceride from closely aligning, which makes them liquid oils at room temperature.

CH3

H3C CH H3C

steroids, such as testosterone and estrogen, function as hormones in multicellular animals. Prostaglandins are a group of about 20 lipids that are modified fatty acids, with two nonpolar “tails” attached to a 5-carbon ring. Prostaglandins act as local chemical messengers in many vertebrate tissues. Chapter 44 explores the effects of some of these complex fatty acids.

CH

CH2 CH2

CH

CH2 CH2

OH

a. Terpene (citronellol) H3C CH3

CH2 CH

CH2 CH2

CH3 CH CH3

CH3

HO

b. Steroid (cholesterol)

Figure 3.29 Other kinds of lipids. a. Terpenes are found in biological pigments, such as chlorophyll and retinal, and (b) steroids play important roles in membranes and as the basis for a class of hormones involved in chemical signaling.

Fats are excellent energy-storage molecules Most fats contain over 40 carbon atoms. The ratio of C—H bonds in fats is more than twice that of carbohydrates (see section 3.2). This means that fats are relatively more reduced than carbohydrates, and will thus release more energy upon oxidation (see chapter 7). This makes fats much more efficient molecules for storing chemical ­energy. On average, fats yield about 9 kilocalories (kcal) of chemical energy per gram, as compared with about 4 kcal/g for carbohydrates. Most fats produced by animals are saturated (except some fish oils), whereas most plant fats are unsaturated (see figure 3.28). The exceptions are the tropical plant oils (palm oil and coconut oil), which are saturated even though they are liquid at room temperature. chapter

3 The Chemical Building Blocks of Life 57

Polar Hydrophilic Heads Nonpolar Hydrophobic Tails

CH2

Figure 3.30  Phospholipids. 

N+(CH3)3

CH2

The phospholipid phosphatidylcholine is shown as (a) a schematic, (b) a formula, (c) a spacefilling model, and (d) an icon used in depictions of biological membranes.

O O P O−

Choline Phosphate Glycerol F a t t y a c i d

H H2C

C

O

O

C O CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C O CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

F a t t y a c i d

a.

O

b.

CH2

c.

d.

When an organism consumes excess carbohydrate, it is converted into starch, glycogen, or fats reserved for future use. The reason that many humans in developed countries gain weight as they grow older is that the amount of energy they need decreases with age, but their intake of food does not. Thus, an increasing proportion of the carbohydrates they ingest is converted into fat. A diet heavy in fats is one of several factors thought to contribute to heart disease, particularly atherosclerosis. In atherosclerosis, sometimes referred to as “hardening of the arteries,” fatty substances called plaque adhere to the lining of blood vessels, blocking the flow of blood. Fragments of a plaque can break off from a deposit and clog arteries to the brain, causing a stroke.

The phospholipid molecule can be thought of as having a polar “head” at one end (the phosphate group) and two long, very nonpolar “tails” at the other (figure 3.30). This structure is essential for how these molecules function, although it first ­appears

Phospholipids form membranes

a.

Complex lipid molecules called phospholipids are among the most important molecules of the cell because they form the core of all biological membranes. An individual phospholipid can be thought of as a substituted triglyceride—that is, a triglyceride with a phosphate replacing one of the fatty acids. The basic structure of a phospholipid includes three kinds of subunits: 1. Glycerol, a 3-carbon alcohol, in which each carbon bears a hydroxyl group. Glycerol forms the backbone of the phospholipid molecule. 2. Fatty acids, long chains of —CH2 groups (hydrocarbon chains) ending in a carboxyl (—COOH) group. Two fatty acids are attached to the glycerol backbone in a phospholipid molecule. 3. A phosphate group (—PO42−) attached to one end of the glycerol. The charged phosphate group usually has a charged organic molecule linked to it, such as choline, ethanolamine, or the amino acid serine. 58 part I The Molecular Basis of Life

Water

Lipid head (hydrophilic) Lipid tail (hydrophobic)

Water

Water

b.

Figure 3.31 Lipids spontaneously form micelles or lipid bilayers in water. In an aqueous environment, lipid molecules

orient so that their polar (hydrophilic) heads are in the polar medium, water, and their nonpolar (hydrophobic) tails are held away from the water. a. Droplets called micelles can form, or (b) phospholipid molecules can arrange themselves into two layers; in both structures, the hydrophilic heads extend outward and the hydrophobic tails inward. This second example is called a phospholipid bilayer.

paradoxical. Why would a molecule need to be soluble in water, but also not soluble in water? The formation of a membrane shows the unique properties of such a structure. In water, the nonpolar tails of nearby lipid molecules aggregate away from the water, forming spherical micelles, with the tails facing inward (figure 3.31a). This is actually how detergent molecules work to make grease soluble in water. The grease is soluble within the nonpolar interior of the micelle and the polar surface of the micelle is soluble in water. With phospholipids, a more complex structure forms in which two layers of molecules line up, with the hydrophobic tails of each layer pointing toward one another, or inward, leaving the hydrophilic heads oriented outward, forming a bilayer (­figure 3.31b). Lipid bilayers are the basic framework of biological membranes, discussed in detail in chapter 5.

Learning Outcomes Review 3.5

Triglycerides are made of fatty acids linked to glycerol. Fats can contain twice as many C—H bonds as carbohydrates and thus they store energy efficiently. Because the C—H bonds in lipids are nonpolar, they are not water-soluble and aggregate together in water. Phospholipids replace one fatty acid with a hydrophilic phosphate group. This allows them to spontaneously form bilayers, which are the basis of biological membranes. ■■

Why do phospholipids form membranes while triglycerides form insoluble droplets?

Chapter Review Deco/Alamy Stock Photo

3.1 Carbon: The Framework

of Biological Molecules

Carbon, the backbone of all biological molecules, can form four covalent bonds and make long chains. Hydrocarbons consist of carbon and hydrogen, and their bonds store considerable energy. Functional groups account for differences in molecular properties. Functional groups are small molecular entities that confer specific chemical characteristics when attached to a hydrocarbon. Carbon and hydrogen have similar electronegativity, so C—H bonds are not polar. Oxygen and nitrogen have greater electronegativity, leading to polar bonds. Isomers have the same molecular formulas but different structures. Structural isomers are molecules with the same formula but different structures; stereoisomers differ in how groups are attached. Enantiomers are mirror-image stereoisomers. Biological macromolecules include carbohydrates, nucleic acids, proteins, and lipids. Most important biological macromolecules are polymers—long chains of monomer units. Biological polymers are formed by elimination of water (H and OH) from two monomers (dehydration reaction). They are broken down by adding water (hydrolysis).

3.2 Carbohydrates: Energy Storage

and Structural Molecules

The empirical formula of a carbohydrate is (CH2O)n. Carbohydrates are used for energy storage and as structural molecules. Monosaccharides are simple sugars. Simple sugars contain three to six or more carbon atoms. Examples are glyceraldehyde (3 carbons), deoxyribose (5 carbons), and glucose (6 carbons).

Sugar isomers have structural differences. The general formula for 6-carbon sugars is C6H12O6, and many isomeric forms are possible. Living systems often have enzymes for converting isomers from one to the other. Disaccharides serve as transport molecules in plants and provide nutrition in animals. Plants convert glucose into the disaccharide sucrose for transport within their bodies. Female mammals produce the disaccharide lactose to nourish their young. Polysaccharides provide energy storage and structural components. Glucose is used to make three important polymers: glycogen (in animals), and starch and cellulose (in plants). Chitin is a related structural material found in arthropods and many fungi.

3.3 Nucleic Acids: Information Molecules Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polymers composed of nucleotide monomers. Cells use nucleic acids for information storage and transfer. Nucleic acid polymers are polynucleotides. Nucleic acids contain four different nucleotide bases. In DNA these are adenine, guanine, cytosine, and thymine. In RNA, thymine is replaced by uracil. DNA stores genetic information. DNA exists as a double helix held together by specific base pairs: adenine with thymine and guanine with cytosine. The nucleic acid sequence constitutes the genetic code. RNA has many roles in a cell. RNA is made by copying DNA. RNA carries information from DNA and forms part of the ribosome. RNA can also be an enzyme and affect gene expression.

chapter

3 The Chemical Building Blocks of Life 59

Denaturation inactivates proteins. Denaturation refers to an unfolding of tertiary structure, which usually destroys function. Some denatured proteins may recover function when conditions are returned to normal. This implies that primary structure strongly influences tertiary structure. Dissociation refers to separation of quaternary subunits with no changes to their tertiary structure.

Other nucleotides are vital components of energy reactions. ATP hydrolysis releases energy that can be used by cells. NAD+ and FAD transport electrons in cellular processes.

3.4 Proteins: Molecules with Diverse Structures

and Functions

Most enzymes are proteins. Proteins also provide defense, transport, motion, and regulation, among many other roles.

3.5 Lipids: Hydrophobic Molecules

Proteins are polymers of amino acids. Amino acids are joined by peptide bonds to make polypeptides. The 20 common amino acids are characterized by R groups that determine their properties.

Lipids are insoluble in water because they have a high proportion of nonpolar C—H bonds. Fats consist of fatty acid polymers attached to glycerol. Many lipids exist as triglycerides, three fatty acids connected to a glycerol molecule. Saturated fatty acids contain the maximum number of hydrogen atoms. Unsaturated fatty acids contain one or more double bonds between carbon atoms.

Proteins have levels of structure. Protein structure is defined by the following hierarchy: primary (amino acid sequence), secondary (hydrogen bonding patterns), tertiary (threedimensional folding), and quaternary (associations between two or more polypeptides).

Fats are excellent energy-storage molecules. Fats have twice the number of C—H bonds as carbohydrates. Thus oxidation yields 9 kcal/g compared with 4 kcal/g. For this reason, excess carbohydrate is converted to fat for storage.

Motifs and domains are structural elements of proteins. Motifs are similar structural elements found in dissimilar proteins. They can create folds, creases, or barrel shapes. Domains are functional subunits or sites within a tertiary structure.

Phospholipids form membranes. Phospholipids contain two fatty acids and one phosphate attached to glycerol. In phospholipid-bilayer membranes, the phosphate heads are hydrophilic and cluster on the two faces of the membrane, and the hydrophobic tails are in the center.

The process of folding relies on chaperone proteins. Chaperone proteins assist in the folding of proteins. Heat shock proteins are an example of chaperone proteins. Improper folding of proteins can result in disease. Some forms of cystic fibrosis are associated with misfolded proteins.

Visual Summary Macromolecules

carbon based

Organic molecules

Polymers

Carbohydrates

Nucleic acids

types

Simple sugars example Glucose

Energy storage

Starch & glycogen

Complex monomers are

Structure

Cellulose & chitin

monomers are

monomers are

Nucleotides

Amino acids

Triglycerides made of

Store information

Levels of structure

Glycerol & 3 fatty acids

RNA

contain

Nitrogenous base Phosphate Ribose sugars

allow for

Primary Secondary Tertiary

Specific chemical properties

types

exhibit

Quaternary

60 part I The Molecular Basis of Life

Lipids

Proteins

function

DNA

Functional groups

Nonpolymers

function Simple sugars

contain

function Energy storage

Phospholipids made of Glycerol, 2 fatty acids & polar head function Membrane formation

Steroids function Membranes and hormones

Review Questions Deco/Alamy Stock Photo

U N D E R S TA N D 1. How is a polymer formed from multiple monomers? a. b. c. d.

From the growth of the chain of carbon atoms By the removal of an —OH group and a hydrogen atom By the addition of an —OH group and a hydrogen atom Through hydrogen bonding

2. Why are carbohydrates important molecules for energy storage? a. The C—H bonds found in carbohydrates store energy. b. The double bonds between carbon and oxygen are very strong. c. The electronegativity of the oxygen atoms means that a carbohydrate is made up of many polar bonds. d. They can form ring structures in the aqueous environment of a cell. 3. Plant cells store energy in the form of _________, and animal cells store energy in the form of ___________. a. b. c. d.

fructose; glucose disaccharides; monosaccharides cellulose; chitin starch; glycogen

4. Which carbohydrate would you find as part of a molecule of RNA? a. b. c. d.

Galactose Deoxyribose Ribose Glucose

5. A molecule of DNA or RNA is a polymer of a. monosaccharides. b. nucleotides.

c. amino acids. d. fatty acids.

6. What makes cellulose different from starch? a.

Starch is produced by plant cells, and cellulose is produced by animal cells. b. Cellulose forms long filaments, and starch is highly branched. c. Starch is insoluble, and cellulose is soluble. d. All of the choices are correct. 7. What monomers make up a protein? a. b. c. d.

Monosaccharides Nucleotides Amino acids Fatty acids

8. A triglyceride is a form of _______ composed of ___________. a. b. c. d.

lipid; fatty acids and glucose lipid; fatty acids and glycerol carbohydrate; fatty acids lipid; cholesterol

A P P LY 1. You can use starch or glycogen as an energy source, but not cellulose because a. b. c. d.

starch and cellulose have similar structures. cellulose and glycogen have similar structures. starch and glycogen have similar structures. your body makes starch but not cellulose.

2. Which of the following is NOT a difference between DNA and RNA? a. b. c. d.

Deoxyribose sugar versus ribose sugar Thymine versus uracil Double-stranded versus single-stranded Phosphodiester versus hydrogen bonds

3. Which part of an amino acid has the greatest influence on the overall structure of a protein? a. b. c. d.

The (—NH2) amino group The R group The (—COOH) carboxyl group Both a and c are correct.

4. A mutation that alters a single amino acid within a protein can alter a. b. c. d.

the primary level of protein structure. the secondary level of protein structure. the tertiary level of protein structure. All of the choices are correct.

5. Two different proteins have the same domain in their structure. From this we can infer that they have a. b. c. d.

the same primary structure. similar function. very different functions. the same primary structure but different functions.

6. What aspect of triglyceride structure accounts for their insolubility in water? a. b. c. d.

The COOH group of fatty acids The nonpolar C—H bonds in fatty acids The OH groups in glycerol The C=C bonds found in unsaturated fatty acids

7. The spontaneous formation of a lipid bilayer in an aqueous environment occurs because a.

the polar head groups of the phospholipids can interact with water. b. the long fatty acid tails of the phospholipids can interact with water. c. the fatty acid tails of the phospholipids are hydrophobic. d. Both a and c are correct.

SYNTHESIZE 1. How do the four biological macromolecules differ from one another? How does the structure of each relate to its function? 2. Hydrogen bonds and hydrophobic interactions each play an important role in stabilizing and organizing biological macromolecules. Consider the four macromolecules discussed in this chapter. Describe how these affect the form and function of each type of macromolecule. Would a disruption in the hydrogen bonds affect form and function? Would hydrophobic interactions do so? 3. Plants make both starch and cellulose. Would you predict that the enzymes involved in starch synthesis could also be used by the plant for cellulose synthesis? Construct an argument to explain this based on the structure and function of the enzymes and the polymers synthesized. chapter

3 The Chemical Building Blocks of Life 61

4

Part II Biology of the Cell

CHAPTER

Cell Structure Chapter Contents 4.1

Cell Theory

4.2

Prokaryotic Cells

4.3

Eukaryotic Cells

4.4

The Endomembrane System

4.5

Mitochondria and Chloroplasts: Cellular Generators

4.6

The Cytoskeleton

4.7

Extracellular Structures and Cell Movement

4.8

Cell-to-Cell Interactions

1 μm Dr. Gopal Murti/Science Source

Introduction

Visual Outline Genetic material Cytoplasm

have have

All cells

have have

Plasma membrane

Ribosomes

Archaea Eukaryotic cells

Prokaryotic cells have

have no

Bacteria

Nucleus have

Flagella

Organelles

surrounded by Nuclear envelope

Cell walls

contain

have

Extracellular structures

have a Cytoskeleton

including

Mitochondria Chloroplasts

Endomembrane system

All organisms are composed of cells. The gossamer wing of a butterfly is a thin sheet of cells and so is the glistening outer layer of your eyes. The burger or tomato you eat is composed of cells, and its contents soon become part of your cells. Some organisms consist of a single cell too small to see with the unaided eye. Others, such as humans, are composed of many specialized cells, such as the fibroblast cell shown in the striking fluorescence micrograph on this page. Cells are so much a part of life that we cannot imagine an organism that is not cellular in nature. In this chapter, we take a close look at the internal structure of cells. In chapters 5 to 10, we will focus on cells in action—how they communicate with their environment, grow, and reproduce.

4.1

Cell Theory

Learning Outcomes 1. Discuss the cell theory. 2. Describe the factors that limit cell size. 3. Categorize structural and functional similarities in cells.

Cells are too small for you to be able to see. Although there are exceptions, a typical eukaryotic cell is 10 to 100 micrometers (μm) in diameter, and most prokaryotic cells are only 1 to 10 μm in diameter. Because cells are so small, they were not discovered until the invention of the microscope in the 17th century. English natural philosopher Robert Hooke was the first to observe cells in 1665, naming the shapes he saw in cork cellulae (Latin, “small rooms”). They are known to us as cells. Another early microscopist, Dutch Anton van Leeuwenhoek, first observed living cells, which he termed “animalcules,” or little animals. After these early efforts, a century and a half passed before biologists fully recognized the importance of cells. In 1838, German botanist Matthias Schleiden stated that all plants “are aggregates of fully individualized, independent, separate beings, namely the cells themselves.” In 1839, German physiologist Theodor Schwann reported that all animal tissues also consist of individual cells. Thus, the cell theory was born.

Cell theory is the unifying foundation of cell biology The cell theory was proposed to explain the observation that all organisms are composed of cells. It sounds simple, but it is a farreaching statement about the organization of life. In its modern form, the cell theory includes the following three principles: 1. All organisms are composed of one or more cells, and the life processes of metabolism and heredity occur within these cells. 2. Cells are the smallest living things, the basic units of organization of all organisms. 3. Cells arise only by division of a previously existing cell. Although life likely evolved spontaneously in the environment of early Earth, biologists have concluded that no additional cells are originating spontaneously at present. Rather, life on Earth represents a continuous line of descent from those early cells.

Cell size is limited Most cells are relatively small for reasons related to the diffusion of substances into and out of them. The rate of diffusion is affected by a number of variables, including (1) surface area available for diffusion, (2) temperature, (3) concentration gradient of diffusing substance, and (4) the distance over which diffusion must occur. As the size of a cell increases, the length of time for diffusion from the outside membrane to the interior of the cell increases as well. Larger cells need to synthesize more macromolecules, have correspondingly higher energy requirements, and produce a greater

Figure 4.1 Surface area-tovolume ratio. As a cell gets larger, its volume increases at a faster rate than its surface area. If the cell radius increases by 10 times, the surface area increases by 100 times, but the volume increases by 1000 times. A cell’s surface area must be large enough to meet the metabolic needs of its volume.

Cell radius (r)

1 unit

10 units

Surface area (4πr 2)

12.57 units2

1257 units2

Volume (4–πr 3)

4.189 units3

4189 units3

3

0.3

3

Surface Area / Volume

quantity of waste. Molecules used for energy and biosynthesis must be transported through the membrane. Any metabolic waste produced must be removed, also passing through the membrane. The rate at which this transport occurs depends on both the distance to the membrane and the area of membrane available. For this reason, an organism made up of many relatively small cells has an advantage over one composed of fewer, larger cells. The advantage of small cell size is readily apparent in terms of the surface area-to-volume ratio. As a cell’s size increases, its volume increases much more rapidly than its surface area. For a spherical cell, the surface area is proportional to the square of the radius, whereas the volume is proportional to the cube of the radius. Thus, if the radii of two cells differ by a factor of 10, the larger cell will have 102, or 100 times, the surface area, but 103, or 1000 times, the volume of the smaller cell (figure 4.1). The cell surface provides the only opportunity for interaction with the environment, because all substances enter and exit a cell via this surface. The membrane surrounding the cell plays a key role in controlling cell function. Because small cells have more surface area per unit of volume than large ones, control over cell contents is more effective when cells are relatively small. Although most cells are small, some quite large cells do exist. These cells have apparently overcome the surface area-to-volume problem by one or more adaptive mechanisms. For example, some cells, such as skeletal muscle cells, have more than one nucleus, allowing genetic information to be spread around a large cell. Some other large cells, such as neurons, are long and skinny, so that any given point within the cell is close to the plasma membrane. This permits diffusion between the inside and outside of the cell to still be rapid.

Microscopes allow visualization of cells and components Other than egg cells, not many cells are visible to the naked eye (figure 4.2). Most are less than 50 µm in diameter, far smaller than the period at the end of this sentence. So, to visualize cells we need chapter

4 Cell Structure 63

100 m

the aid of technology. The development of microscopes and their refinement over the centuries has allowed us to continually explore cells in greater detail.

10 m

The resolution problem Human Eye

1m

Adult human

10 cm

1 cm

1 mm

Light Microscope

100 μm

10 μm

Chicken egg

Frog egg Paramecium

Human egg

Human red blood cell

Electron Microscope

Prokaryote

1 μm

100 nm

Chloroplast Mitochondrion

Large virus (HIV)

Ribosome 10 nm Protein

1 nm

0.1 nm (1 Å)

Amino acid

Hydrogen atom

Logarithmic scale

Figure 4.2 The size of cells and their contents. Except for vertebrate eggs, which can typically be seen with the unaided eye, most cells are microscopic in size. Prokaryotic cells are generally 1 to 10 µm across. 1 m = 102 cm = 103 mm = 106 µm = 109 nm 64 part II Biology of the Cell

How do we study cells if they are too small to see? The key is to understand why we can’t see them. The reason we can’t see such small objects is the limited resolution of the human eye. Resolution is the minimum distance two points can be apart and still be distinguished as two separate points. When two objects are closer together than about 100 µm, the light reflected from each strikes the same photoreceptor cell at the rear of the eye. Only when the objects are farther than 100 µm apart can the light from each strike different cells, allowing your eye to resolve them as two distinct objects rather than one.

Types of microscopes One way to overcome the limitations of our eyes is to increase magnification so that small objects appear larger. The first microscopists used glass lenses to magnify small cells and cause them to appear larger than the 100-µm limit imposed by the human eye. The glass lens increases focusing power. Because the glass lens makes the object appear closer, the image on the back of the eye is bigger than it would be without the lens. Modern light microscopes, which operate with visible light, use two magnifying lenses (and a variety of correcting lenses) to achieve very high magnification and clarity (­table 4.1). The first lens focuses the image of the object on the second lens, which magnifies it again and focuses it on the back of the eye. Microscopes that magnify in stages using several lenses are called compound microscopes. They can resolve structures that are separated by at least 200 nanometers (nm). Light microscopes, even compound ones, are not powerful enough to resolve many of the structures within cells. For example, a cell membrane is only 5 nm thick. Why not just add another magnifying stage to the microscope to increase its resolving power? This doesn’t work because when two objects are closer than a few hundred nanometers, the light beams reflecting from the two images start to overlap each other. The only way two light beams can get closer together and still be resolved is if their wavelengths are shorter. One way to avoid overlap is by using a beam of electrons rather than a beam of light. Electrons have a much shorter wavelength, and an electron microscope, employing electron beams, has 1000 times the resolving power of a light microscope. Transmission electron microscopes, so called because the electrons are transmitted through a specimen to visualize it, can resolve objects only 0.2 nm apart—which is only twice the diameter of a hydrogen atom! A second kind of electron microscope, the scanning ­electron microscope, beams electrons onto the surface of the specimen. The electrons reflected back from the surface, together with other electrons that the specimen itself emits as a result of the bombardment, are amplified and transmitted to a screen, where the image can be viewed and photographed. Scanning electron microscopy yields striking three-­ dimensional images. This technique has improved our understanding of many biological and physical phenomena (table 4.1).

Using stains to view cell structure Although resolution remains a physical limit, we can improve the images we see by altering the sample. Certain chemical stains

TA B L E 4 .1

Microscopes

LIGHT MICROSCOPES Bright-field microscope: Light is transmitted through a specimen, giving little contrast. Staining specimens improves contrast but requires that cells be fixed (not alive), which can distort or alter components. Nancy Nehring/E+/

Getty Images

Dark-field microscope: Light is directed at an angle toward the specimen. A condenser lens transmits only light reflected off the specimen. The field is dark, and the specimen is light against this dark background. Laguna Design/ Science Source

28 μm 28 μm

68 μm 68 μm

Phase-contrast microscope: Components of the microscope bring light waves out of phase, which produces differences in contrast and brightness when the light waves recombine. De Agostini Picture Library/age fotostock

Differential-interference–contrast microscope: Polarized light is split into two beams that have slightly different paths through the sample. Combining these two beams produces greater contrast, especially at the edges of structures.

All cells share simple structural features 33 μm 33 μm

27 μm 27 μm

Source

10 μm 10 μm

Confocal microscope: Light from a laser is focused to a point and scanned across the fluorescently stained specimen in two directions. This produces clear images of one plane of the specimen. Other planes of the specimen are excluded to prevent the blurring of the image. Multiple planes can be used to reconstruct a 3-D image. C.J. Guerin, PhD, MRC Toxicology Unit/ Science Source

ELECTRON MICROSCOPES

IMAGES/Science Source

Scanning electron microscope: An electron beam is scanned across the surface of the specimen, and electrons are knocked off the surface. Thus, the topography of the specimen determines the contrast and the content of the image. False coloring enhances the image. Steve Gschmeissner/Science Source

Every cell contains DNA for genetic material. In prokaryotes, the simplest organisms, this consists primarily of a single circular molecule of DNA. It is typically found near the center of the cell in an area called the nucleoid. This region is not surrounded by a membrane. By contrast, eukaryotic cells are more complex, with DNA organized into linear chromosomes segregated into a nucleus, which is surrounded by a double-membrane structure called the nuclear envelope (described in section 4.3). In all cells, the DNA contains the genes that code for the proteins synthesized by the cell.

The cytoplasm

25 μm 25 μm

Transmission electron microscope: A beam of electrons is passed through the specimen. Electrons that pass through are used to expose film. Areas of the specimen that scatter electrons appear dark. False coloring enhances the image. AMI

The general plan of cellular organization varies between different organisms, but despite these modifications, all cells resemble one another in certain fundamental ways. Before we begin a detailed examination of cell structure, let’s first summarize four major features all cells have in common: (1) a nucleoid or nucleus where genetic material is located, (2) cytoplasm, (3) ­ribosomes to synthesize proteins, and (4) a plasma membrane.

Centrally located genetic material

F. Fox/picture alliance/blickwinkel/F/Newscom

Fluorescence microscope: Fluorescent stains absorb light at one wavelength, then emit it at another. Filters transmit only the emitted light. Dr. Torsten Wittmann/Science

increase the contrast between different cellular components. Structures within the cell absorb or exclude the stain differentially, producing contrast that aids resolution. Stains that bind to specific types of molecules have made these techniques even more powerful. This method uses antibodies that bind, for example, to a particular protein. This process, called immunohistochemistry, uses antibodies generated in animals such as rabbits or mice. When these animals are injected with specific proteins, they produce antibodies that bind to the injected protein. The antibodies are then purified and chemically bonded to enzymes, to stains, or to fluorescent molecules. When cells are incubated in a solution containing the antibodies, the antibodies bind to cellular structures that contain the target molecule and can be seen with light microscopy. This approach has been used extensively in the analysis of cell structure and function.

A semifluid matrix called the cytoplasm fills the interior of the cell. The cytoplasm contains all of the sugars, amino acids, and proteins the cell uses to carry out its everyday activities. Although it is an aqueous medium, cytoplasm is more like Jell-O than water due to the high concentration of proteins and other macromolecules. The fluid part of the cytoplasm, which contains organic molecules and ions in solution, is called the cytosol. Membrane-bound organelles, usually specialized for a particular function and containing specific proteins, create compartments in the cytoplasm.

Ribosomes 3 μm 3 μm

Ribosomes are large, macromolecular machines composed of RNA and protein that synthesize all cellular proteins. Ribosomes use information from the genome and other accessory molecule to direct the synthesis of proteins by sequential addition of amino acids.

The plasma membrane 7 μm 7 μm

The plasma membrane encloses a cell and separates its contents from its surroundings. The plasma membrane is a phospholipid bilayer about 5 to 10 nm (5 to 10 billionths of a meter) thick, with proteins embedded in it. Viewed in cross section with the electron chapter

4 Cell Structure 65

Prokaryotic cells are more complex than we thought

Protein Plasma membrane

Cell interior 20 nm

Don W. Fawcett/Science Source

microscope, membranes appear as two dark lines separated by a lighter area. This arises from the tail-to-tail organization of phospholipid molecules that make up the membrane (see chapter 5). The proteins of the plasma membrane are generally responsible for a cell’s ability to interact with the environment. Membrane proteins give cells identity, and provide for a variety of functions, including transport and communication with other cells and the environment. The role of the plasma membrane is so important, we will study it in detail in chapter 5.

Learning Outcomes Review 4.1

All organisms are single cells or aggregates of cells, and all cells arise from preexisting cells. Cell size is limited primarily by the efficiency of diffusion across the plasma membrane. As a cell becomes larger, its volume increases more quickly than its surface area. Past a certain point, diffusion cannot support the cell’s needs. All cells are bounded by a plasma membrane and filled with cytoplasm. The genetic material is found in the central portion of the cell; and in eukaryotic cells, it is contained in a membrane-bounded nucleus. ■■

When cells were visualized with microscopes, two basic cellular architectures were recognized: eukaryotic and prokaryotic. These terms refer to the presence or absence, respectively, of a membranebounded nucleus that contains genetic material. In addition to lacking a nucleus, prokaryotic cells do not have an internal membrane system or numerous membrane-bounded organelles. Our view of prokaryotic cell structure has changed significantly over the years. They have been described as small, simple cells consisting of a plasma membrane containing cytoplasm with genetic material and ribosomes, and encased by a rigid cell wall (figure 4.3). While accurate as far as it goes, this is an oversimplification.

?

Inquiry question What modifications would you include if you wanted to make a cell as large as possible?

Prokaryotes were thought to lack any membrane-bounded organelles, which form compartments in eukaryotes. While still accurate to say that there are no organelles common to all prokaryotes, particular species do have specific organelles that provide specialized functions. The best studied of these is the magnetosome, found in bacteria that can move along a magnetic field. Magnetosomes consist of spherical membranes containing iron oxide crystals.

Pilus

Would finding life on Mars change our view of cell theory?

4.2 Prokaryotic

Figure 4.3  Structure of a prokaryotic cell.  Generalized cell

Cells

Cytoplasm organization of a Ribosomes Nucleoid (DNA)

Learning Outcomes 1. Explain how our view of prokaryotic cell structure has changed. 2. Distinguish between bacterial and archaeal cell types.

Prokaryotes are very important in the ecology of living systems. Some harvest light by photosynthesis, others break down dead organisms and recycle their components. Still others cause disease or are used in many industrial processes. Prokaryotes have two main domains: Archaea and Bacteria, introduced here and discussed in detail in chapter 27.

Plasma membrane Cell wall Capsule

prokaryote. The nucleoid is visible as a dense central region segregated from the cytoplasm. Some prokaryotes have hairlike growths (called pili [singular, pilus]) on the outside of the cell. 

Biology Pics/Science Source

Pili

Flagellum

0.3 μm

66 part II Biology of the Cell

Antibiotic drugs used to combat bacterial infection often target their cell walls. For example, penicillin and vancomycin interfere with the ability of bacteria to cross-link the peptides in their peptidoglycan cell wall. Like removing all the nails from a wooden house, this destroys the integrity of the structural matrix, which can no longer prevent water from rushing in and swelling the cell to bursting. Some bacteria also secrete a jellylike protective capsule of polysaccharide around the cell. Many disease-causing bacteria have such a capsule, which enables them to adhere to teeth, skin, food—or practically any surface that can support their growth. Fe3O4

Figure 4.4 Electron micrograph of magnetosomes. 

Magnetosomes are membrane-enclosed spheres containing iron oxide crystals. These allow magnetotactic bacteria to align with a magnetic field.  DENNIS KUNKEL MICROSCOPY/Science Source

These are arrayed in a chain by attachment to an external fiber, which allows the cell to align with a magnetic field (figure 4.4). Both bacterial and archaeal species may also have internal membranebound storage structures. Prokaryotes can also have infoldings of the plasma membrane that serve to segregate metabolic reactions. Some bacteria also contain cellular compartments bounded by a semipermeable protein shell. These bacterial microcompartments (BMCs) range in size from 40 to 400 nm, and act to isolate a specific metabolic process, or to store a particular substance. While the structure is quite different, these compartments are functional analogs of eukaryotic organelles. Prokaryotes were also thought to lack the elaborate cytoskeleton found in eukaryotes, but we have now found they have molecules related to both actin and tubulin, which form two of the cytoskeletal elements described in section 4.6. The strength and shape of the cell is determined by the cell wall and not these cytoskeletal elements (figure 4.3). However, cell wall structure is influenced by the cytoskeleton. For instance, the presence of actin-like MreB fibers running the length of the cell leads to perpendicular cell-wall fibers that produce a rod-shaped cell. This can be seen when MreB protein is removed: cells become spherical rather than rod-shaped. During cell division, cell-wall deposition is influenced by the tubulin-like FtsZ protein (see chapter 10).

Bacterial cell walls consist of peptidoglycan Most bacterial cells are encased by a strong cell wall composed of peptidoglycan, which consists of a carbohydrate matrix crosslinked with short polypeptide units (see chapter 27 for details). Cell walls protect the cell, maintain its shape, and prevent excessive uptake or loss of water. The exception is the class Mollicutes, which includes the common genus Mycoplasma, which lack a cell wall. Plants, fungi, and most protists also have cell walls but with a chemical structure different from that of peptidoglycan.

Archaea have unusual membrane lipids We have much to learn about the physiology and structure of archaea. Many archaeans are difficult to culture in the laboratory, as shown recently by the decade-long effort required to culture a single deep sea archaean. It is much easier to identify archaeal genomes in environmental samples, so we know more about archaeal genomics than about archaeal physiology. The cell walls of archaea are more diverse than bacteria, containing polysaccharides and proteins, and possibly even inorganic components. One feature that distinguishes archaea from bacteria is the structure of their membrane lipids. Archaeal membrane lipids include saturated hydrocarbons that are covalently attached to glycerol at both ends, which forms a monolayer membrane (see chapter 27 for details). These features seem to confer greater thermal stability to archaeal membranes, although the trade-off seems to be an inability to alter the degree of saturation of the hydrocarbons—limiting their response to changing environmental temperatures. The cellular machinery that replicates DNA and synthesizes proteins in archaea is more closely related to eukaryotic systems than to bacterial systems. Even though they share a similar overall cellular architecture with bacteria, archaea appear to be more closely related on a molecular basis to eukaryotes.

Bacterial and archaeal flagellar structures evolved independently Bacterial, archaeal, and eukaryotic cells all have external structures for motility—long filaments made of proteins—that superficially appear similar, but actually are three evolutionarily distinct structures. Bacterial flagella (singular flagellum) consists of protein rings embedded in the plasma membrane, and a cell wall with long protein fibers extending from this (figure 4.5a). The analogous archaeal structure, now called an archaellum, consists of a structure that appears related to a different bacterial external structure known as a pilus (see chapter 27). This is formed by a disk of membrane proteins with protein filaments extending from the cell (figure 4.5b). Both bacterial and archaeal structures rotate like a propellor, while eukaryotes have whip-like flagella. However, the motive force for rotation is different: bacteria use a proton gradient similar to that used in eukaryotic mitochondria (see chapter 7), while archaea use the hydrolysis of ATP. The independent evolution of different structures with similar function is called convergent evolution, discussed in more detail in chapter 21. chapter

4 Cell Structure 67

Hook

Figure 4.5 Comparison of bacterial flagellum and archaellum. a. The bacterial

Filament

Outer membrane Peptidoglycan portion of cell wall

S layer H+

H+

Plasma membrane

ATP

Plasma membrane

ADP

a.

Flagellum

b.

Learning Outcomes Review 4.2

The two domains of prokaryotes are archaea and bacteria. Some species have membrane-bounded organelles, and semipermeable protein compartments. Bacteria cell walls are composed of peptidoglycan, while archaea have cell walls made from a variety of polysaccharides and peptides, as well as membranes containing unusual lipids. Bacteria and archaea have rotating motility structures that evolved independently. ■■

What features do bacteria and archaea share?

flagellum consists of concentric discs embedded in the membrane and cell wall. These are connected to a protein fiber composed of subunits of flagellin. The structure rotates powered by the flow of protons into the cell. b. The archaellum, found in all motile archaea, is built on components related to those in a type of bacterial pilus. In this case, rotation of the protein fiber is powered by the hydrolysis of ATP.

Archaellum

sacs that store and transport a variety of materials. Inside the nucleus, the DNA is wound tightly around proteins and packaged into compact units called chromosomes. All eukaryotic cells are supported by an internal protein scaffold, the cytoskeleton. Although the cells of animals and some protists lack cell walls, the cells of fungi, plants, and many protists have strong cell walls composed of cellulose or chitin fibers embedded in a matrix of other polysaccharides and proteins. Through the rest of this chapter, we will examine the internal components of eukaryotic cells in more detail.

The nucleus acts as the information center

4.3 Eukaryotic

Cells

Learning Outcomes 1. Compare the organization of eukaryotic and prokaryotic cells. 2. Discuss the role of the nucleus in eukaryotic cells. 3. Describe the role of ribosomes in protein synthesis.

Eukaryotic cells (figures 4.6 and 4.7) are far more complex than prokaryotic cells. The hallmark of the eukaryotic cell is compartmentalization. This is achieved through a combination of an extensive endomembrane system that weaves through the cell interior and by numerous organelles. These organelles include membrane-bounded structures that form compartments within which multiple biochemical processes can proceed simultaneously and independently. Plant cells often have a large, membrane-bounded sac called a central vacuole, which stores proteins, pigments, and waste materials. Both plant and animal cells contain vesicles—smaller 68 part II Biology of the Cell

The largest and most easily seen organelle within a eukaryotic cell is the nucleus (Latin, “kernel” or “nut”), first described by the Scottish botanist Robert Brown in 1831. Nuclei are roughly spherical in shape, and in animal cells, they are typically located in the central region of the cell (figure 4.8a). In some cells, a network of fine cytoplasmic filaments seems to cradle the nucleus in this position. The nucleus contains the genetic information that enables the synthesis of nearly all proteins of a living eukaryotic cell. Most eukaryotic cells possess a single nucleus, although the cells of fungi and some other groups may have from several to many nuclei. Mammalian erythrocytes (red blood cells) lose their nuclei when they mature. Many nuclei exhibit a dark-staining zone called the n ­ ucleolus, which is a region where intensive synthesis of ribosomal RNA is taking place.

The nuclear envelope The surface of the nucleus is bounded by two phospholipid bilayer membranes, which together make up the nuclear ­envelope (figure 4.8). The outer membrane of the nuclear envelope is continuous with the cytoplasm’s interior membrane system, called the endoplasmic reticulum (described in section 4.4).

Figure 4.6 Structure of an animal cell. In this generalized diagram of an animal cell, the plasma

membrane encases the cell, which contains the cytoskeleton and various cell organelles and interior structures suspended in a semifluid matrix called the cytoplasm. Some kinds of animal cells possess fingerlike projections called microvilli. Other types of eukaryotic cells—for example, many protist cells—may possess flagella, which aid in movement, or cilia, which can have many different functions.

Nucleus

Ribosomes

Nuclear envelope

Rough endoplasmic reticulum

Nucleolus

Smooth endoplasmic reticulum

Nuclear pore Intermediate filament

Microvilli

Cytoskeleton Actin filament (microfilament) Microtubule

Ribosomes

Intermediate filament

Centriole Cytoplasm Lysosome

Exocytosis Vesicle Golgi apparatus

Plasma membrane Peroxisome

Mitochondrion

chapter

4 Cell Structure 69

Figure 4.7 Structure of a plant cell. Most mature plant cells contain a large central vacuole, which

occupies a major portion of the internal volume of the cell, and organelles called chloroplasts, within which photosynthesis takes place. The cells of plants, fungi, and some protists have cell walls, although the composition of the walls varies among the groups. Plant cells have cytoplasmic connections to one another through openings in the cell wall called plasmodesmata. Flagella occur in sperm of a few plant species, but are otherwise absent from plant and fungal cells. Centrioles are also usually absent.

Rough endoplasmic reticulum

Nucleus

Smooth endoplasmic reticulum

Nuclear envelope

Ribosome

Nuclear pore Nucleolus Intermediate filament Central vacuole Cytoskeleton Intermediate filament Microtubule Actin filament (microfilament) Peroxisome Mitochondrion

Golgi apparatus

Cytoplasm

Vesicle Chloroplast

Adjacent cell wall Cell wall Plasma membrane

70 part II Biology of the Cell

Plasmodesmata

Figure 4.8 The nucleus. a. The nucleus is composed of a double membrane called the nuclear envelope, enclosing a fluid-filled interior containing chromatin. The individual nuclear pores extend through the two membrane layers of the envelope. The close-up of the nuclear pore shows the central hub, cytoplasmic ring with fibers, and nuclear ring with basket. b. A freeze-fracture electron micrograph (see figure 5.4) of a cell nucleus, showing many nuclear pores. c. A transmission electron micrograph of the nuclear membrane showing a single nuclear pore. The dark material within the pore is protein, which acts to control access through the pore. d. The nuclear lamina is visible as a dense network of fibers made of intermediate filaments. The nucleus has been colored purple in the micrographs.  (b) Don W. Fawcett/Science

Nuclear pores Nuclear envelope Nucleolus Chromatin Nucleoplasm Nuclear lamina

Source; (c) Don W. Fawcett/Science Source; (d) Dr. Ueli Aebi

Scattered over the surface of the nuclear envelope are what appear as shallow depressions in the electron micrograph but are in fact structures called nuclear pores (figure 4.8b, c). These pores form 50 to 80 nm apart at locations where the two membrane layers of the nuclear envelope come together. The structure consists of a central framework with eightfold symmetry that is embedded in the nuclear envelope. This is bounded by a cytoplasmic face with eight fibers, and a nuclear face with a complex ring that forms a basket beneath the central ring. The pore allows ions and small molecules to diffuse freely between nucleoplasm and cytoplasm, while controlling the passage of proteins and RNA–protein complexes. Transport across the pore is controlled and consists mainly of the import of proteins that function in the nucleus, and the export to the cytoplasm of RNA and RNA–protein complexes formed in the nucleus. The inner surface of the nuclear envelope is covered with a network of fibers that make up the nuclear lamina (figure 4.8d). This is composed of intermediate filament fibers called nuclear lamins. This structure gives the nucleus its shape and is also involved in the deconstruction and reconstruction of the nuclear envelope that accompanies cell division.

Chromatin: DNA packaging In both prokaryotes and eukaryotes, DNA is the molecule that stores genetic information. In eukaryotes, the DNA is divided into multiple linear chromosomes, which are organized with proteins into a complex structure called chromatin. It is becoming clear that the very structure of chromatin affects the function of DNA. Changes in gene expression that do not involve changes in DNA sequence, so-called epigenetic changes, involve alterations in chromatin structure (see chapter 16). Although still not fully understood, this offers an exciting new view of many old ideas. Chromatin is also more organized in the nucleus than was once thought. The state of the DNA in chromatin must also change over the course of cell division, as we will see in chapter 10. Chromosomes become compacted into a more highly condensed state that forms the X-shaped chromosomes visible in the light microscope.

The nucleolus: Ribosomal subunit manufacturing Before cells can synthesize proteins in large quantity, they must first construct a large number of ribosomes to carry out this

Inner membrane

Nuclear basket

Outer membrane Cytoplasmic filaments

a.

Nuclear pore Nuclear pores

Cytoplasm

Pore

Nucleus

b.

300 nm

c.

d.

150 nm

1 μm

synthesis. Hundreds of copies of the genes encoding the ribosomal RNAs are clustered together on the chromosome, facilitating ribosome construction. By transcribing RNA molecules from this cluster, the cell rapidly generates large numbers of the molecules needed to produce ribosomes. The clusters of ribosomal RNA genes, the RNAs they produce, and the ribosomal proteins all come together within the chapter

4 Cell Structure 71

Large subunit

Ribosome Small subunit

Figure 4.9 A ribosome. Ribosomes consist of a large and a

small subunit composed of rRNA and protein. The individual subunits are synthesized in the nucleolus and then move through the nuclear pores to the cytoplasm, where they assemble to translate mRNA. Ribosomes serve as sites of protein synthesis.

nucleus during ribosome production. These ribosomal assembly areas are easily visible within the nucleus as one or more darkstaining regions called nucleoli (singular, nucleolus). Nucleoli can be seen under the light microscope even when the chromosomes are uncoiled.

Ribosomes are the cell’s protein synthesis machinery Although the DNA in a cell’s nucleus encodes the amino acid sequence of each protein in the cell, the proteins are not assembled there. A simple experiment demonstrates this: if a brief pulse of radioactive amino acid is administered to a cell, the radioactivity shows up associated with newly made protein in the cytoplasm, not in the nucleus. When investigators first carried out these experiments, they found that protein s­ynthesis is associated with large RNA– protein complexes (called ribosomes) outside the nucleus. Ribosomes are among the most complex molecular assemblies found in cells. Each ribosome is composed of two subunits (figure 4.9), each of which is composed of a combination of RNA, called ribosomal RNA (rRNA), and proteins. The subunits join to form a functional ribosome only when they are actively synthesizing proteins. This complicated process requires the two other main forms of RNA: messenger RNA (mRNA), which carries coding information from DNA, and transfer RNA (tRNA), which carries amino acids. Ribosomes use the information in mRNA to direct the synthesis of a protein. This process will be described in more detail in chapter 15. Ribosomes are found either free in the cytoplasm or associated with internal membranes, as described in section 4.4. Free ribosomes synthesize proteins that are found in the cytoplasm, nuclear proteins, mitochondrial proteins, and proteins found in other organelles not derived from the endomembrane system. Membrane-associated ribosomes synthesize membrane proteins, proteins found in the endomembrane system, and proteins destined for export from the cell. Ribosomes can be thought of as “universal organelles” because they are found in all cell types from all three domains of life. As we build a picture of the minimal essential functions for cellular life, ribosomes will be on the short list. Life is p­ rotein-based, and ribosomes are the factories that make proteins. 72 part II Biology of the Cell

Learning Outcomes Review 4.3

In contrast to prokaryotic cells, eukaryotic cells exhibit compartmentalization. Eukaryotic cells contain an endomembrane system and organelles that carry out specialized functions. The nucleus, composed of a double membrane connected to the endomembrane system, contains the cell’s genetic information. Material moves between the nucleus and cytoplasm through nuclear pores. Ribosomes translate mRNA, which is transcribed from DNA in the nucleus, into polypeptides that make up proteins. Ribosomes are a universal organelle found in all known cells. ■■

Would you expect transport through nuclear pores to be a regulated process?

4.4 The Endomembrane System Learning Outcomes 1. Identify the different parts of the endomembrane system. 2. Contrast the different functions of internal membranes and compartments. 3. Evaluate the importance of each step in the proteinprocessing pathway.

The interior of a eukaryotic cell is packed with membranes that form an elaborate internal, or endomembrane, system. This endo­ membrane system fills the cell, dividing it into compartments, channeling the passage of molecules through the interior of the cell, and providing surfaces for the synthesis of lipids and some proteins. The endomembrane system in eukaryotic cells is one of the fundamental distinctions between eukaryotes and prokaryotes. The endoplasmic reticulum (ER) is the largest internal membrane. The ER is composed of a phospho­lipid bilayer embedded with proteins. The ER has functional subdivisions, described here, and forms a variety of structures, from folded sheets to complex tubular networks (figure 4.10). The ER may also be connected to the cytoskeleton, which can affect ER structure and growth. The two largest compartments in eukary­otic cells are the space inside the ER, called the cisternal space, or lumen, and the region exterior to it, the cytosol, which is the fluid component of the cytoplasm containing dissolved organic molecules and ions.

The rough ER is a site of protein synthesis The rough ER (RER) gets its name from the pebbly appearance of its surface. The RER is not easily visible with a light microscope, but it can be seen using the electron microscope. It appears to consist primarily of flattened sacs with surfaces made bumpy by ribosomes (figure 4.10). The proteins synthesized on the surface of the RER are destined to be exported from the cell, sent to lysosomes or vacu­oles (described later in this section), or embedded in the plasma membrane.

Ribosomes Rough endoplasmic reticulum

Figure 4.10  The endoplasmic reticulum. Rough ER

(RER), blue in the drawing, is composed more of flattened sacs and forms a compartment throughout the cytoplasm. Ribosomes associated with the cytoplasmic face of the RER extrude newly made proteins into the interior, or lumen. The smooth ER (SER), green in the drawing, is a more tubelike structure connected to the RER. The micrograph has been colored to match the drawing.  Don W. Fawcett/

Smooth endoplasmic reticulum

Rough endoplasmic reticulum

Smooth endoplasmic reticulum 80 nm

The ratio of SER to RER is not fixed but depends on a cell’s function. In multicellular animals, like humans, this ratio can vary greatly. Cells that carry out extensive lipid synthe­sis, such as those in the testes, intestine, and brain, have abundant SER. Cells that synthesize secreted proteins, for example, anti­bodies, have much more extensive RER. Another role of the SER is to modify foreign substances to make them less toxic. In the liver, enzymes in the SER carry out this detoxification. This can remove substances that we have taken for a therapeutic reason, for instance, penicillin. Thus, relatively high doses of some drugs are prescribed to offset our body’s efforts to remove them. Liver cells have extensive SER with enzymes that can process a variety of substances by chemically modifying them.

The Golgi apparatus sorts and packages proteins Flattened stacks of membranes form a complex called the Golgi body, or Golgi apparatus (figure 4.11). These structures are named for Camillo Golgi, the 19th-century Italian physician who first identified them. The individual stacks of membrane are called cisternae (Latin, “collecting vessels”), and they vary in number within the Golgi body from 1 or a few in protists to 20 or more in animal cells, and to several hundred in plant cells. In vertebrates, individual Golgi are linked to form a Golgi ribbon. They are espe­ cially abundant in glandular cells, which manufacture and secrete substances.

Science Source

These proteins enter the cisternal space as a first step in the pathway that will sort proteins to their eventual destinations. This pathway also involves vesicles and the Golgi apparatus. The sequence of the protein being synthesized determines whether the ribosome will become associated with the ER or remain a cytoplasmic ribosome. In the ER, newly synthesized proteins can be modified by the addition of short-chain carbohydrates to form glycoproteins. Those proteins bound for secretion are separated from other products and packaged into vesicles that move to the Golgi for further modification and packaging for export.

Transport vesicle

cis face Fusing vesicle

Forming vesicle trans face Secretory vesicle

The smooth ER has multiple roles Regions of the ER with relatively few bound ribosomes are referred to as smooth ER (SER). The structure of the SER ranges from a network of tubules, to flattened sacs, to higher-order tubular arrays. The membranes of the SER contain many embedded enzymes, involved in the synthesis of a variety of carbohy­drates and lipids. Steroid hormones are also synthesized in the SER. The majority of membrane lipids are assembled in the SER and then sent to whatever parts of the cell need membrane components. Membrane proteins in the plasma membrane and other cellular membranes are inserted by ribo­somes on the RER. An important function of the SER is to store intracellular Ca2+. This keeps the cytoplasmic level low, allowing Ca2+ to be used as a signaling molecule. In muscle cells, for example, Ca2+ is used to trigger muscle contraction. In other cells, Ca2+ release from SER stores is involved in diverse signaling pathways.

1 μm

Figure 4.11  The Golgi apparatus. The Golgi apparatus is a

smooth, concave, membranous structure. It receives material for processing in transport vesicles on the cis face and sends the material packaged in transport or secretory vesicles off the trans face. The substance in a vesicle could be for export out of the cell or for distribution to another region within the same cell.  Dennis Kunkel

Microscopy/Science Source

chapter

4 Cell Structure 73

Nucleus Nuclear pore

Ribosome

Rough endoplasmic reticulum Membrane protein Newly synthesized protein 1. Vesicle containing proteins buds from the rough endoplasmic reticulum, diffuses through the cell, and fuses to the cis face of the Golgi apparatus.

Transport vesicle Smooth endoplasmic reticulum

cis face

Golgi membrane protein

Cisternae

The Golgi apparatus functions in the collection, packaging, and distribution of molecules synthesized at one location and used at another within the cell or even outside of it. A Golgi body has a front and a back, each with a distinct membrane composition. The receiving side is called the cis face and is usually located near the ER. Materials arrive at the cis face in transport vesicles that bud off the ER and exit the trans face, where they are discharged in secretory vesicles (figure 4.12). How material transits through the Golgi has been a source of contention. Models include maturation of the individual cisternae from cis to trans, transport between cisternae by vesicles, and direct tubular connections. Although there is prob­ably transport of material by all of these, it now appears that the primary mechanism is cisternal maturation. Proteins and lipids manufactured on the rough and smooth ER membranes are transported into the Golgi apparatus and modi­ fied as they pass through it. The most common alteration is the addition or modification of short sugar chains, forming glycopro­ teins and glycolipids. In many instances, enzymes in the Golgi apparatus modify existing glycoproteins and glycolipids made in the ER by cleaving a sugar from a chain or by modifying one or more of the sugars. These are then packaged into vesicles that pinch off from the trans face of the Golgi. These vesicles then diffuse to other locations in the cell, distributing the newly synthesized molecules to their appropriate destinations. Another function of the Golgi apparatus is the synthesis of cell-wall components. Noncellulose polysaccharides that form part of the cell wall of plants are synthesized in the Golgi apparatus and sent to the plasma membrane, where they can be added to the cellulose that is assembled on the exterior of the cell. Other polysaccharides secreted by plants are also synthesized in the Golgi apparatus.

Lysosomes contain digestive enzymes Golgi Apparatus trans face

2. The proteins are modified and packaged into vesicles for transport.

Secretory vesicle

Secreted protein

Cell membrane

3. The vesicle may travel to the plasma membrane, releasing its contents to the extracellular environment.

Extracellular fluid

Figure 4.12 Protein transport through the endomembrane system. Proteins synthesized by ribosomes on

the RER are translocated into the internal compartment of the ER. These proteins may be used at a distant location within the cell or secreted from the cell. They are transported within vesicles that bud off the RER. These transport vesicles travel to the cis face of the Golgi apparatus. There they can be modified and packaged into vesicles that bud off the trans face of the Golgi apparatus. Vesicles leaving the trans face transport proteins to other locations in the cell, or fuse with the plasma membrane, releasing their contents to the extracellular environment.

74 part II Biology of the Cell

Lysosomes are digestive vesicles that arise from the Golgi apparatus. They contain high levels of a variety of enzymes that can degrade proteins, nucleic acids, lipids, and carbohydrates. As these enzymes could destroy the cell, it is critical to segregate them. Throughout the lives of eukaryotic cells, lysosomal enzymes break down old organelles and recycle their component molecules, allowing room for newly formed organelles. For example, mitochondria are replaced in some tissues every 10 days. The digestive enzymes in the lysosome are optimally active at acid pH. Lysosomes are activated by fusing with a food vesicle produced by phagocytosis (see chapter 5) or by fusing with an old or worn-out organelle. The fusion event activates proton pumps in the lysosomal membrane, lowering the internal pH. As the interior pH falls, the digestive enzymes contained in the lysosome become active. This leads to the degradation of macromolecules in the food vesicle or the destruction of the old organelle. A number of human genetic disorders, collectively called lysosomal storage disorders, affect lysosomes. For example, Tay– Sachs disease is caused by the loss of function of a single lysosomal enzyme (hexosaminidase). This enzyme is necessary to break down a membrane glycolipid found in nerve cells. Accumulation of glycolipid in lysosomes affects nerve cell function, leading to a variety of clinical symptoms such as seizures and muscle rigidity. In addition to breaking down organelles and other structures within cells, lysosomes eliminate other cells that the cell has engulfed

by phagocytosis. When a white blood cell, for example, phagocytoses a passing pathogen, lysosomes fuse with the resulting “food vesicle,” releasing their enzymes into the vesicle and degrading the material within (figure 4.13).

Nucleus

Nuclear pore

Ribosome

Rough endoplasmic reticulum

Lipid droplets store neutral lipids Lipid droplets are organelles with a different structure from the rest of the endomembrane system. They consist of a neutral lipid core surrounded by a single layer of phospholipid. Neutral lipids are hydrophobic and include triglycerides and sterol lipids. These neutral lipids are made soluble in the aqueous cytosol by being coated with a phospholipid. Lipid droplets vary in size in different cells, and over time. They form storage depots for lipids that are used for energy metabolism, and to form membranes. Lipid droplets can also contain proteins, or have proteins on the surface, involved in lipid synthesis. This allows them to act as a secondary site of lipid synthesis. Thus these organelles form a hub for both energy and membrane metabolism. Their size will vary with the needs of the cell, growing and shrinking as lipids are added or used.

Microbodies are a diverse category of organelles

Membrane protein

Eukaryotic cells have a variety of vesicles called microbodies that contain enzymes. These are found in the cells of plants, animals, fungi, and protists. The distribution of enzymes into microbodies is one of the principal ways eukaryotic cells orga­nize their metabolism.

Hydrolytic enzyme

Peroxisomes: Peroxide utilization

Transport vesicle cis face

Golgi membrane protein Smooth endoplasmic reticulum

Peroxisomes (figure 4.14) are microbodies that contain enzymes used to oxidize fatty acids. If these oxidative enzymes were free in the cytoplasm, they could short-circuit cellular metabolism, which

Cisternae

Golgi Apparatus

trans face

Lysosome

Old or damaged organelle

Lysosome

Breakdown of organelle Lysosome aiding in the breakdown of an old organelle

Digestion

Food vesicle

Phagocytosis Lysosome aiding in the digestion of phagocytized particles

Figure 4.13 Lysosomes. Lysosomes are formed from vesicles

budding off the Golgi. They contain hydrolytic enzymes that digest particles or cells taken into the cell by phagocytosis, and break down old organelles.

0.2 μm

Figure 4.14 A peroxisome. Peroxisomes are spherical organelles that may contain a large crystal structure composed of protein. Peroxisomes contain digestive and detoxifying enzymes that produce hydrogen peroxide as a by-product. A peroxisome has been colored green in the electron micrograph.  Don W. Fawcett/Science Source chapter

4 Cell Structure 75

often involves adding hydrogen atoms to oxygen. Peroxisomes get their name from the hydrogen peroxide produced as a by-product of the activities of oxidative enzymes. Hydrogen peroxide is dangerous to cells because of its violent chemical reactivity. However, peroxisomes also contain the enzyme catalase, which catalyzes the decomposition of hydrogen peroxide into water and oxygen. Because many peroxisomal proteins are synthesized by cytoplasmic ribosomes, the organelles themselves were long thought to form by the addition of lipids and proteins, leading to growth. As they grow larger, they divide to produce new peroxisomes. Although division of peroxisomes still appears to occur, it is now clear that peroxisomes can form from the fusion of ER-derived vesicles. These vesicles then import peroxisomal proteins to form a mature peroxisome. Genetic screens have isolated some 32 genes that encode proteins involved in biogenesis and maintenance of peroxisomes. The human genetic diseases called peroxisome biogenesis disorders (PBDs) can be caused by mutations in some of these genes.

Plants use vacuoles for storage and water balance Plant cells have specialized membrane-bounded structures called vacuoles. The most conspicuous example is the large central vacu­ ole seen in most plant cells (figure 4.15). In fact, vacuole actually means blank space, referring to its appearance in the light micro­ scope. The membrane surrounding this vacuole is called the tonoplast because it contains channels for water that are used to help the cell maintain its tonicity, or osmotic balance (see section 5.4). For many years biologists assumed there was only one type of vacuole with multiple functions. These included water balance and storage of useful molecules (sugars, ions, and pigments) and waste products. The vacuole was also thought to contain enzymes used to break down macromolecules, and to detoxify foreign substances.

Some referred to vacuoles as the attic of the cell for the variety of substances thought to be stored there. Studies of tonoplast transporters and the isolation of vacu­ oles from a variety of cell types has led to a more complex view of vacuoles. These studies make it clear that different vacuo­lar types are found in different cells. These vacuoles are specialized, depending on the function of the cell. The central vacuole is clearly important for a number of roles in all plant cells. The water channels in the tonoplast maintain the tonicity of the cell, allowing the cell to expand and contract, depending on conditions. The central vac­uole is also involved in cell growth by occupying most of the vol­ume of the cell. Plant cells grow by expanding the vacuole, rather than by increasing cytoplasmic volume. Vacuoles with a variety of functions are also found in some fungi and protists. One form is the contractile vacuole, found in some protists, which can pump water to main­tain water balance in the cell. Other vacuoles are used for storage or to segregate toxic materials from the rest of the cytoplasm. The number and kind of vacuoles found in a cell depend on the needs of the particular cell type.

Learning Outcomes Review 4.4

The endoplasmic reticulum (ER) is an extensive system of membranes that spatially organize the cell’s biosynthetic activities. Lipid and membrane synthesis occurs on smooth ER, which also stores Ca2+. Rough ER (RER) is covered with ribosomes that synthesize proteins, which can be transported by vesicles to the Golgi apparatus, where they are modified, packaged, and distributed to their final location. Lysosomes contain digestive enzymes used to degrade materials such as invaders or worn-out organelles. Lipid droplets store neutral lipids and contain enzymes. Peroxisomes carry out oxidative metabolism that generates peroxides. Vacuoles are membrane-bounded structures with roles ranging from storage to cell growth in plants. They are also found in some fungi and protists. ■■

How do ribosomes on the RER differ from cytoplasmic ribosomes?

4.5 Mitochondria

and Chloroplasts: Cellular Generators

Nucleus Central vacuole

Learning Outcomes

Tonoplast Chloroplast

Cell wall

1. Describe the structure of mitochondria and chloroplasts. 2. Compare the function of mitochondria and chloroplasts. 3. Explain the probable origin of mitochondria and chloroplasts. 1.5 μm

Figure 4.15 The central vacuole. A plant’s central vacuole

stores dissolved substances and can expand in size to increase the tonicity of a plant cell. Micrograph shown with false color.  Biophoto Associates/

Science Source

76 part II Biology of the Cell

Mitochondria and chloroplasts share structural and functional similarities. Structurally, they are both surrounded by a double membrane, and both contain their own DNA and protein synthesis machinery. Functionally, they are both involved in energy

Ribosome Matrix Crista

DNA

division are encoded by genes in the nucleus and are translated into proteins by cytoplasmic ribosomes. Mitochondrial replication is, therefore, impossible without nuclear participation, and mitochondria thus cannot be grown in a cell-free culture.

Chloroplasts use light to generate ATP and sugars

Intermembrane space Inner membrane Outer membrane

0.2 μm

Figure 4.16 Mitochondria. The inner membrane of a

mitochondrion is shaped into folds called cristae that greatly increase the surface area for oxidative metabolism. A mitochondrion in cross section and cut lengthwise is shown colored red in the micrograph. 

Keith R.Porter/Science Source

Plant cells and cells of other eukaryotic organisms that carry out photosynthesis typically contain from one to several hundred chloroplasts. Chloroplasts bestow an obvious advantage on the organisms that possess them: they can manufacture their own food. Chloroplasts contain the photosynthetic pigment chlorophyll that gives most plants their green color. The chloroplast, like the mitochondrion, is surrounded by two membranes (figure 4.17). However, chloroplasts are larger and more complex than mitochondria. In addition to the outer and inner membranes, which lie in close association with each other, chloroplasts have closed compartments of stacked membranes called grana (singular, granum), which lie inside the inner membrane. A chloroplast may contain a hundred or more grana, and each granum may contain from a few to several dozen disk-shaped structures called thylakoids. On the surface of the thylakoids are the light-capturing photosynthetic pigments, to be discussed in depth in chapter 8. Surrounding the thylakoid is a fluid matrix called the stroma. The enzymes used to synthesize glucose during photosynthesis are found in the stroma.

metabolism, as we will explore in detail in chapters 7 and 8, on energy metabolism and photosynthesis. Ribosome

Mitochondria generate most cellular ATP Mitochondria (singular, mitochondrion) vary in both size and shape, often existing as part of a dynamic network, and are found in all types of eukaryotic cells (figure 4.16). Mitochondria are bounded by two membranes: a smooth outer membrane, and an inner folded membrane with numerous contiguous layers called cristae (singular, crista). The cristae partition the mitochondrion into two compartments: a matrix, lying inside the inner membrane; and an outer compartment, or intermembrane space, lying between the two mitochondrial membranes. On the surface of the inner membrane, and also embedded within it, are proteins that carry out oxidative metabolism, the oxygen-requiring process by which energy in macromolecules is used to produce ATP (see chapter 7). Mitochondria have their own DNA; this DNA contains several genes that produce proteins essential to the mitochondrion’s role in oxidative metabolism. Thus, the mitochondrion, in many respects, acts as a cell within a cell, containing its own genetic information specifying proteins for its unique functions. The mitochondria are not fully autonomous, however, because nearly all of the genes that encode the enzymes used in oxidative metabolism are located in the cell nucleus. A eukaryotic cell does not produce brand-new mitochondria each time the cell divides. Instead, the mitochondria themselves divide in two, doubling in number, and these are partitioned between the new cells. Most of the components required for mitochondrial

DNA

Thylakoid membrane Outer membrane Inner membrane Thylakoid disk

Granum

Stroma

Stroma Granum

0.5 μm

Figure 4.17 Chloroplast structure. The inner membrane of a chloroplast surrounds a membrane system of stacks of closed chlorophyll-containing vesicles called thylakoids, within which photosynthesis occurs. Thylakoids are typically stacked one on top of the other in columns called grana. The chloroplast has been colored green in the micrograph. Jeremy Burgess/Science Source chapter

4 Cell Structure 77

Like mitochondria, chloroplasts contain DNA, but many of the genes that specify chloroplast components are also located in the nucleus. Some of the elements used in photosynthesis, including the specific protein components necessary to accomplish the reaction, are synthesized entirely within the chloroplast. Other DNA-containing organelles in plants, called leucoplasts, lack pigment and a complex internal structure. In root cells and some other plant cells, leucoplasts may serve as starch-storage sites. A leucoplast that stores starch (amylose) is sometimes termed an amyloplast. These organelles—chloroplasts, leucoplasts, and amyloplasts—are collectively called plastids. All plastids are produced by the division of existing plastids.

?

Inquiry question Mitochondria and chloroplasts both generate ATP. What structural features do they share?

Mitochondria and chloroplasts arose by endosymbiosis Symbiosis is a close relationship between organisms of different species that live together. The theory of endosymbiosis proposes that some of today’s eukaryotic organelles evolved by a symbiosis arising between two cells that were each free-living. One cell, a prokaryote, was engulfed by and became part of another cell, which was the precursor of modern eukaryotes (figure 4.18). According to the endosymbiont theory, the engulfed prokaryotes provided their hosts with certain advantages associated with their special metabolic abilities. Two key eukaryotic organelles are believed to be the descendants of these endosymbiotic prokaryotes: mitochondria, which are thought to have originated as bacteria capable of carrying out oxidative metabolism, and chloroplasts, which apparently arose from photosynthetic bacteria. This is discussed in detail in chapter 28.

Learning Outcomes Review 4.5

Nucleus Unknown Archaean

Proteobacterium

Mitochondrion Chloroplast

Cyanobacterium

Mitochondria and chloroplasts have similar structures, with an outer membrane and an extensive inner membrane compartment. Both mitochondria and chloroplasts have their own DNA, but both also depend on nuclear genes for some functions. Mitochondria and chloroplasts are both involved in energy conversion: mitochondria metabolize sugar to produce ATP, whereas chloroplasts harness light energy to produce ATP and synthesize sugars. Endosymbiosis theory proposes that both mitochondria and chloroplasts arose as prokaryotic cells were engulfed by a eukaryotic precursor. ■■

Many proteins in mitochondria and chloroplasts are encoded by nuclear genes. In light of the endosymbiont hypothesis, how might this come about?

Modern Eukaryote

4.6 The Unknown Bacterium

Nucleus

Unknown Archaean Mitochondrion Proteobacterium

Cyanobacterium

Chloroplast

Modern Eukaryote

Figure 4.18 Possible origins of eukaryotic cells. Both

mitochondria and chloroplasts are thought to have arisen by endosymbiosis when a free-living cell was taken up but not digested. The nature of the engulfing cell is unknown. Two possibilities are (1) the engulfing cell (top) was an archaean that gave rise to the nuclear genome and cytoplasmic contents; and (2) the engulfing cell (bottom) consisted of a nucleus derived from an archaean in a bacterial cell. This could have arisen by a fusion event or by engulfment of the archaean by the bacterium.

78 part II Biology of the Cell

Cytoskeleton

Learning Outcomes 1. Contrast the structure and function of different fibers in the cytoskeleton. 2. Illustrate the role of microtubules in intracellular transport.

The cytoplasm of all eukaryotic cells is crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. This network, called the cytoskeleton, is a dynamic system, constantly assembling and disassembling. Individual fibers consist of polymers of identical protein subunits that attract one another and spontaneously assemble into long chains. Fibers disassemble in the same way, as one subunit after another breaks away from one end of the chain.

Three types of fibers compose the cytoskeleton Eukaryotic cells may contain the following three types of cytoskeletal fibers, each formed from a different kind of subunit: (1) actin

filaments, sometimes called microfilaments; (2) microtubules; and (3) intermediate filaments.

Actin filaments (microfilaments) Actin filaments are long fibers about 7 nm in diameter. Each filament is composed of two protein chains loosely twined together like two strands of pearls (figure 4.19). Each “pearl,” or subunit, on the chain is the globular protein actin. Actin filaments exhibit polarity—that is, they have plus (+) and minus (−) ends. These designate the direction of growth of the filaments. Actin molecules spontaneously form these filaments, even in a test tube. Cells regulate the rate of actin polymerization through other proteins that act as switches, turning on polymerization when appropriate. Actin filaments are responsible for cellular movements such as contraction, crawling, “pinching” during division, and formation of cellular extensions.

Microtubule

Intermediate filament Actin filament Cell membrane

Microtubules Microtubules, the largest of the cytoskeletal elements, are hollow tubes about 25 nm in diameter, each composed of a ring of 13 protein protofilaments (figure 4.19). Globular proteins consisting of dimers of α- and β-tubulin subunits polymerize to form the 13 protofilaments. The protofilaments are arrayed side by side around a central core, giving the microtubule its characteristic tube shape. In many cells, microtubules form from nucleation centers near the center of the cell and radiate toward the periphery. They are in a constant state of flux, continually polymerizing and depolymerizing. The average half-life of a microtubule ranges from as long as 10 minutes in a nondividing animal cell to as short as 20 seconds in a dividing animal cell. The ends of the microtubule are designated as plus (+) (away from the nucleation center) or minus (−) (toward the nucleation center). Along with facilitating cellular movement, microtubules organize the cytoplasm and are responsible for moving materials within the cell itself, as described shortly.

Intermediate filaments The most durable element of the cytoskeleton in animal cells is a system of tough, fibrous protein molecules twined together in an overlapping arrangement (figure 4.19). These intermediate filaments are characteristically 8 to 10 nm in diameter—between the size of actin filaments and that of microtubules. Once formed, intermediate filaments are stable and usually do not break down. Intermediate filaments constitute a mixed group of cytoskeletal fibers. The most common type, composed of protein subunits called vimentin, provides structural stability for many kinds of cells. Keratin, another class of intermediate filament, is found in epithelial cells (cells that line organs and body cavities) and associated structures such as hair and fingernails. The intermediate filaments of nerve cells are called neurofilaments.

Centrosomes are microtubule-organizing centers Centrioles are barrel-shaped organelles found in the cells of animals and most protists. They occur in pairs, usually located at right angles to each other near the nuclear membranes (­figure 4.20). The region surrounding the pair in almost all animal cells is referred to as a centrosome. Surrounding the centrioles in the centrosome is

a. Actin filaments

b. Microtubules

c. Intermediate filaments

Figure 4.19 Molecules that make up the cytoskeleton. 

a. Actin filaments: Actin filaments, also called microfilaments, are made of two strands of the globular protein actin twisted together. They are often found in bundles or in a branching network. Actin filaments in many cells are concentrated below the plasma membrane in bundles known as stress fibers, which may have a contractile function. b. Microtubules: Microtubules are composed of α- and β-tubulin protein subunits arranged side by side to form a tube. Microtubules are comparatively stiff cytoskeletal elements and have many functions in the cell including intracellular transport and the separation of chromosomes during mitosis. c. Intermediate filaments: Intermediate filaments are composed of overlapping staggered tetramers of protein. These tetramers are then bundled into cables. This molecular arrangement allows for a ropelike structure that imparts tremendous mechanical strength to the cell.

the pericentriolar material, which contains ring-shaped structures composed of tubulin. The pericentriolar material can nucleate the assembly of microtubules in animal cells. Structures with this function are called microtubule-organizing centers. The centrosome is also responsible for the reorganization of microtubules that occurs during cell division. The centrosomes of plants and fungi lack centrioles, but still contain microtubule-organizing centers. You will learn more about the actions of the centrosomes when we describe the process of cell division in chapter 10. chapter

4 Cell Structure 79

Vesicle

Dynactin complex

Dynein

Microtubule triplet

Figure 4.20 Centrioles. Each centriole is composed of nine triplets of microtubules. Centrioles are usually not found in plant cells. In animal cells they help to organize microtubules.

The cytoskeleton helps move materials within cells Actin filaments and microtubules often orchestrate their activities to affect cellular processes. For example, during cell reproduction (see chapter 10), newly replicated chromosomes move to opposite sides of a dividing cell because they are attached to shortening microtubules. Then, in animal cells, a belt of actin pinches the cell in two by contracting like a purse string. Muscle cells also use actin filaments, which slide along filaments of the motor protein myosin when a muscle contracts. The fluttering of an eyelash, the flight of an eagle, and the awkward crawling of a baby all depend on these cytoskeletal movements within muscle cells. Not only is the cytoskeleton responsible for the cell’s shape and movement, but it also provides a scaffold that interacts with the ER and other cytoplasmic macromolecules. Enzymes involved in cell metabolism bind to actin filaments, as do ribosomes. By moving and anchoring particular enzymes near one another, the cytoskeleton helps organize the cell’s activities. In both animals and fungi, the ER is associated with both microtubules and actin filaments. In animal cells, if we visualize cellular structures with fluorescent labels, the tubules of the ER align closely with microtubules. This may be involved in the growth and distribution of the ER. In yeast, microfilaments are involved in inheritance of the ER during cell division.

Microtubule

Figure 4.21  Molecular motors. 

Vesicles can be transported along microtubules using motor proteins that use ATP to generate force. The vesicles are attached to motor proteins by connector molecules, such as the dynactin complex shown here. The motor protein dynein moves the connected vesicle along microtubules.

uses ATP to power its movement toward the cell periphery, dragging the vesicle with it as it travels along the microtubule toward the plus end (figure 4.22). As nature’s tiniest motors, these proteins pull the transport vesicles along the microtubular tracks. Another set of vesicle proteins, called the dynactin complex, binds vesicles to the motor protein dynein (figure 4.22), which directs movement in the opposite direction along microtubules toward the minus end, inward toward the cell’s center. (Dynein is also involved in the movement of eukaryotic flagella, as discussed in section 4.7.) The destination of a particular transport vesicle and its contents is thus determined by the nature of the linking protein embedded within the vesicle’s membrane. The major eukaryotic cell structures and their respective functions are summarized in table 4.2. SCIENTIFIC THINKING Hypothesis: Kinesin molecules can act as molecular motors and move along microtubules using energy from ATP. Test: A microscope slide is covered with purified kinesin. Purified microtubules are added in a buffer containing ATP. The microtubules are monitored under a microscope using a video recorder to capture any movement.

Molecular motors All eukaryotic cells must move materials from one place to another in the cytoplasm. One way cells do this is by using the channels of the ER as an intracellular highway. Material can also be moved using vesicles loaded with cargo that can move along the cytoskeleton like a railroad track. For example, in a nerve cell with an axon that may extend far from the cell body, vesicles can be moved along tracks of microtubules from the cell body to the end of the axon. Four components are required to move material along microtubules: (1) a vesicle or organelle that is to be transported, (2) a motor protein that provides the energy-driven motion, (3) a connector molecule that connects the vesicle to the motor molecule, and (4) microtubules on which the vesicle will ride like a train on a rail (figure 4.21). The direction a vesicle is moved depends on the type of motor protein involved and the fact that microtubules are organized with their plus ends toward the periphery of the cell. In one case, a protein called kinectin binds vesicles to the motor protein kinesin. Kinesin 80 part II Biology of the Cell

Frame 1

Frame 2

Frame 3

Result: Over time, the movement of individual microtubules can be observed in the microscope. This is shown schematically in the figure by the movement of specific microtubules shown in color. Conclusion: Kinesin acts as a molecular motor moving along (in this case actually moving) microtubules. Further Experiments: Are there any further controls that are not shown in this experiment? What additional conclusions could be drawn by varying the amount of kinesin sticking to the slide?

Figure 4.22 Demonstration of kinesin as molecular motor. Microtubules can be observed moving over a slide coated

with kinesin.

TA B L E 4 . 2 Structure

Eukaryotic Cell Structures and Their Functions Description

Function

Plasma membrane

Phospholipid bilayer with embedded proteins

Regulates what passes into and out of cell; cell-to-cell recognition; connection and adhesion; cell communication

Nucleus

Structure (usually spherical) that contains chromosomes and is surrounded by double membrane

Instructions for protein synthesis and cell reproduction; contains genetic information

Chromosomes

Long threads of DNA that form a complex with protein

Contain hereditary information used to direct synthesis of proteins

Nucleolus

Site of genes for rRNA synthesis

Synthesis of rRNA and ribosome assembly

Ribosomes

Small, complex assemblies of protein and RNA, often bound to ER

Sites of protein synthesis

Endoplasmic reticulum (ER)

Network of internal membranes

Intracellular compartment forms transport vesicles; participates in lipid synthesis and synthesis of membrane or secreted proteins

Golgi apparatus

Stacks of flattened vesicles

Packages proteins for export from cell; forms secretory vesicles

Lysosomes

Vesicles derived from Golgi apparatus that contain hydrolytic digestive enzymes

Digest worn-out organelles and cell debris; digest material taken up by endocytosis

Microbodies

Vesicles that are formed from incorporation of lipids and proteins and that contain oxidative and other enzymes

Isolate particular chemical activities from rest of cell

Mitochondria

Bacteria-like elements with double membrane

“Power plants” of the cell; sites of oxidative metabolism

Chloroplasts

Bacteria-like elements with double membrane surrounding a third, thylakoid membrane containing chlorophyll, a photosynthetic pigment

Sites of photosynthesis

Cytoskeleton

Network of protein filaments

Structural support; cell movement; movement of vesicles within cells

Flagella (cilia)

Cellular extensions with 9 + 2 arrangement of pairs of microtubules

Motility or moving fluids over surfaces

Cell wall

Outer layer of cellulose or chitin; or absent

Protection; support

chapter

4 Cell Structure 81

Learning Outcomes Review 4.6

The three principal fibers of the cytoskeleton are actin filaments (microfilaments), microtubules, and intermediate filaments. These fibers interact to modulate cell shape and permit cell movement. They also act to move materials within the cytoplasm. Material is also moved in large cells using vesicles and molecular motors. The motor proteins move vesicles along tracks of microtubules. ■■

What advantage does the cytoskeleton give to large eukaryotic cells?

4.7 Extracellular

Structures and Cell Movement

Learning Outcomes 1. Describe how cells move. 2. Identify the different cytoskeletal elements involved in cell movement. 3. Classify the elements of extracellular matrix in animal cells.

Essentially all cell motion is tied to the movement of actin filaments, microtubules, or both. Intermediate filaments act as intracellular tendons, preventing excessive stretching of cells. Actin filaments play a major role in determining the shape of cells. Because actin filaments can form and dissolve so readily, they enable some cells to change shape quickly.

Some cells crawl The arrangement of actin filaments within the cell cytoplasm allows cells to crawl, literally! Crawling is a significant cellular phenomenon, essential to such diverse processes as inflammation, clotting, wound healing, and the spread of cancer. White blood cells in particular exhibit this ability. Produced in the bone marrow, these cells are released into the circulatory system and then eventually crawl out of venules and into the tissues to destroy potential pathogens. At the leading edge of a crawling cell, actin filaments rapidly polymerize, and their extension forces the edge of the cell forward. This extended region is stabilized when microtubules polymerize into the newly formed region. Overall forward movement of the cell is then achieved through the action of the protein myosin, which is best known for its role in muscle contraction. Myosin motors along the actin filaments contract, pulling the contents of the cell toward the newly extended front edge. Cells crawl when these steps occur continuously, with a leading edge extending and stabilizing, and then motors contracting to pull the remaining cell contents along. Receptors on the cell surface can detect molecules outside the cell and stimulate extension in specific directions, allowing cells to move toward particular targets. 82 part II Biology of the Cell

Flagella and cilia aid movement In section 4.2, we saw that bacterial and archaeal flagella evolved independently, and now we see a third kind of flagella evolved in eukaryotes: the eukaryotic flagellum, consisting of a circle of nine microtubule pairs surrounding two central microtubules. This arrangement is referred to as the 9 + 2 structure (figure 4.23). As pairs of microtubules move past each other using arms composed of the motor protein dynein, the eukaryotic flagellum undulates, or waves up and down, rather than rotates. When examined carefully, each flagellum proves to be an outward projection of the cell’s interior, containing cytoplasm and enclosed by the plasma membrane. The microtubules of the flagellum are derived from a basal body, situated just below the point where the flagellum protrudes from the surface of the cell. The flagellum’s complex microtubular apparatus evolved early in the history of eukaryotes. Today the cells of many multicellular and some unicellular eukaryotes no longer possess flagella and are nonmotile. Other structures, called cilia (singular, cilium), with an organization similar to the 9 + 2 arrangement of microtubules, can still be found within them. Cilia are short cellular projections that are often organized in rows. They are more numerous than flagella on the cell surface, but have the same internal structure. In many multicellular organisms, cilia carry out tasks far removed from their original function of propelling cells through water. In several kinds of vertebrate tissues, for example, the beating of rows of cilia moves water over the tissue surface.

Doublet microtubule Flagellum

Radial spoke Dynein arm Plasma membrane Basal body

Central microtubule pair

0.1 μm

Microtubule triplet 0.1 μm

Figure 4.23 Flagella and cilia. A eukaryotic flagellum originates directly from a basal body. The flagellum has two microtubules in its core connected by radial spokes to an outer ring of nine paired microtubules with dynein arms (9 + 2 structure). The basal body consists of nine microtubule triplets connected by short protein segments. The structure of cilia is similar to that of flagella, but cilia are usually shorter.  Dr. William Dentler/University of Kansas

Plasmodesmata

Primary wall

Secondary wall Plant cell Plasma membrane

Middle lamella

Cell 2

a.

40 μm Primary wall Secondary wall

Cell 1 Middle lamella

Plasma membrane

b.

67 μm

Figure 4.24 Flagella and cilia. a. A green alga with numerous flagella that allow it to move through the water. b. Paramecia are covered with many cilia, which beat in unison to move the cell. The cilia can also be used to move fluid into the paramecium’s mouth to ingest material.  SPL/Science Source

0.4 μm

Figure 4.25 Cell walls in plants. Plant cell walls are thick, strong, and rigid. Primary cell walls are laid down when the cell is young. Thicker secondary cell walls may be added later when the cell is fully grown.  Biophoto Associates/Science Source

Animal cells secrete an extracellular matrix The sensory cells of the vertebrate ear also contain conventional cilia surrounded by actin-based stereocilia; sound waves bend these structures and provide the initial sensory input for hearing. Thus, the 9 + 2 structure of flagella and cilia appears to be a fundamental component of eukaryotic cells (figure 4.24).

Plant cell walls provide protection and support The cells of plants, fungi, and many types of protists have cell walls, which protect and support the cells. The cell walls of these eukaryotes are chemically and structurally different from prokaryotic cell walls. In plants and protists, the cell walls are composed of fibers of the polysaccharide cellulose, whereas in fungi, the cell walls are composed of chitin. In plants, primary walls are laid down when the cell is still growing. Between the walls of adjacent cells a sticky substance, called the middle lamella, glues the cells together (figure 4.25). Some plant cells produce strong secondary walls, which are deposited inside the primary walls of fully expanded cells.

Animal cells lack the cell walls that encase plants, fungi, and most protists. Instead, animal cells secrete an elaborate mixture of glycoproteins into the space around them, forming the extracellular matrix (ECM) (figure 4.26). The fibrous protein collagen, the same protein found in cartilage, tendons, and ligaments, may be abundant in the ECM. Strong fibers of collagen and another fibrous protein, elastin, are embedded within a complex web of other glycoproteins, called proteoglycans, that form a protective layer over the cell surface.

?

Inquiry question The passageways of the human

trachea (the path of air flow into and out of the lungs) are known to be lined with ciliated cells. What function could these cilia perform?

The ECM of some cells is attached to the plasma membrane by a third kind of glycoprotein, fibronectin. Fibronectin molecules bind not only to ECM glycoproteins but also to proteins called integrins. Integrins are an integral part of the plasma membrane, extending into the cytoplasm, where they are attached to the microfilaments and intermediate filaments of the cytoskeleton. chapter

4 Cell Structure 83

Collagen

Linking ECM and cytoskeleton, integrins allow the ECM to influence cell behavior in important ways. They can alter gene expression and cell migration patterns by a combination of mechanical and chemical signaling pathways. In this way, the ECM can help coordinate the behavior of all the cells in a particular tissue. Table 4.3 compares and reviews the features of three types of cells.

Elastin

Fibronectin Integrin

Proteoglycan

Learning Outcomes Review 4.7

Cell movement involves proteins. These can either be internal in the case of crawling cells that use actin and myosin, or external in the case of cells powered by cilia or flagella. Eukaryotic cilia and flagella are different from prokaryotic flagella because they are composed of bundles of microtubules in a 9 + 2 array. They undulate rather than rotate.

Actin filament

Plant cells have a cellulose-based cell wall. Animal cells lack a cell wall. In animal cells, the cytoskeleton is linked to a web of glycoproteins called the extracellular matrix.

Cytoplasm

■■

Figure 4.26 The extracellular matrix. Animal cells are

surrounded by an extracellular matrix composed of various glycoproteins that give the cells support, strength, and resilience.

TA B L E 4 . 3

What cellular roles are performed by microtubules and microfilaments and not intermediate filaments?

A Comparison of Prokaryotic, Animal, and Plant Cells Prokaryote

Animal

Plant

EXTERIOR STRUCTURES Cell wall

Present (protein-polysaccharide)

Absent

Present (cellulose)

Cell membrane

Present

Present

Present

Flagella/cilia

Flagella may be present

May be present (9 + 2 structure)

Absent except in sperm of a few species (9 + 2 structure)

INTERIOR STRUCTURES Endoplasmic reticulum

Absent

Usually present

Usually present

Ribosomes

Present

Present

Present

Microtubules

Absent

Present

Present

Centrioles

Absent

Present

Absent

Golgi apparatus

Absent

Present

Present

Nucleus

Absent

Present

Present

Mitochondria

Absent

Present

Present

Chloroplasts

Absent

Absent

Present

Chromosomes

Single; circle of DNA

Multiple; DNA–protein complex

Multiple; DNA–protein complex

Lysosomes

Absent

Usually present

Present

Vacuoles

Absent

Absent or small

Usually a large single vacuole

84 part II Biology of the Cell

4.8 Cell-to-Cell

Interactions

Learning Outcomes 1. Describe the roles of surface proteins. 2. Differentiate between types of cell junctions.

A basic feature of multicellular animals is the formation of diverse kinds of tissue, such as skin, blood, or muscle, where cells are organized in specific ways. Cells must also be able to communicate with each other and have markers of individual identity. All of these functions—connections between cells, markers of cellular identity, and cell communication—involve membrane proteins and proteins secreted by cells. As an organism develops, the cells acquire their identities by carefully controlling the expression of those genes, turning on the specific set of genes that encodes the functions of each cell type. Table 4.4 provides a summary of the kinds of connections seen between cells that are explored in this section.

Surface proteins give cells identity One key set of genes functions to mark the surfaces of cells, identifying them as being of a particular type. When cells make contact, they “read” each other’s cell-surface markers and react accordingly. Cells that are part of the same tissue type recognize each other, and they frequently respond by forming connections between their surfaces to better coordinate their functions.

TA B L E 4 . 4 Type of Connection

Glycolipids Most tissue-specific cell-surface markers are glycolipids—that is, lipids with carbohydrate heads. The glycolipids on the surface of red blood cells are also responsible for the A, B, and O blood types.

MHC proteins One example of the function of cell-surface markers is the recognition of “self ” and “nonself ” cells by the immune system. This function is vital for multicellular organisms, which need to defend themselves against invading or malignant cells. The immune system of vertebrates uses a particular set of markers to distinguish self from nonself cells, encoded by genes of the major histocompatibility complex (MHC). Cell recognition in the immune system is covered in chapter 50.

Cell connections mediate cell-to-cell adhesion The evolution of multicellularity required the acquisition of molecules that can connect cells to each other. It appears that multicellularity arose independently in different lineages, but the types of connections between cells are remarkably conserved, and many of the proteins involved are ancient. The nature of the physical connections between the cells of a tissue in large measure determines what the tissue is like. Indeed, a tissue’s proper functioning often depends critically on how the individual cells are arranged within it. Just as a house cannot maintain its structure without nails and cement, so a tissue cannot maintain its characteristic architecture without the appropriate cell junctions. Cell junctions can be characterized by both their visible structure in the microscope, and the proteins involved in the junction.

Cell-to-Cell Connections and Cell Identity Structure

Function

Example

Surface markers

Variable, integral proteins or glycolipids in plasma membrane

Identify the cell

MHC complexes, blood groups, antibodies

Septate junctions

Tightly bound, leakproof, fibrous claudin protein seal that surrounds cell

Hold cells together such that materials pass through but not between the cells

Junctions between epithelial cells in the gut

Adhesive junction (desmosome)

Variant cadherins, desmocollins, bind to intermediate filaments of cytoskeleton

Creates strong flexible connections between cells. Found in vertebrates

Epithelium

Adhesive junction (adherens junction)

Classical cadherins, bind to microfilaments of cytoskeleton

Connects cells together. Oldest form of cell junction, found in all multicellular organisms

Tissues with high mechanical stress, such as the skin

Adhesive junction (hemidesmosome, focal adhesion)

Integrin proteins bind cell to extracellular matrix

Provides attachment to a substrate

Involved in cell movement and important during development

Communicating junction (gap junction)

Six transmembrane connexon/pannexin proteins creating a pore that connects cells

Allows passage of small molecules from cell to Excitable tissue such as heart muscle cell in a tissue

Communicating junction (plasmodesmata)

Cytoplasmic connections between gaps in adjoining plant cell walls

Communicating junction between plant cells

Tight junctions

Plant tissues

chapter

4 Cell Structure 85

Adhesive junctions Adhesive junctions appear to have been the first to evolve. They mechanically attach the cytoskeleton of a cell to the cytoskeletons of other cells or to the ECM. These junctions are found in tissues subject to mechanical stress, such as muscle and skin epithelium. Adherens junctions are formed by cadherin molecules on the surface of cells. The cadherin superfamily is a large family of Ca2+-dependent adhesion molecules found in virtually all meta­ zoan animals. The first cadherin molecules appeared more than 600 million years ago in the last common ancestor to all animals, and have expanded over time. There are 114 members of this superfamily in the human genome. Given the ancient origin of cadherins, it is not surprising that adherans junctions are found in all metazoan animals, even in Placazoans, which are essentially a sheet of cells, and Cnidarians (see chapter 32). Cadherin is a single-pass transmembrane protein with an extracellular domain that can interact with the extracellular domain of a cadherin in an adjacent cell to join the cells together (figure 4.27). Cadherins found in these junctions are called classical cadherins, which we divide into types I and II. When cells bearing either type I or type II cadherins are mixed, they sort into populations joined by I-to-I or by II-to-II interactions. There is some interaction between type I and type II cadherins, but they are not as strong. On the cytoplasmic side, the cadherins interact indirectly through other proteins with actin to form flexible connections between cells (figure 4.27). Desmosomes are a cadherin-based junction unique to vertebrates. They contain the cadherins desmocollin and desmoglein, which interact with intermediate filaments of cytoskeletons instead of actin. Desmosomes join adjacent cells (figure 4.28b). These connections support tissues against mechanical stress.

Cytoplasm

β

NH2

Adjoining cell membrane

Extracellular domains of cadherin protein

Cadherin of adjoining cell

Intercellular space

Cytoplasm

A Ac ct ctin Actin

Plasma membrane

β α γ x

COOH CO COO H In nttra r Intracellular att a tac a attachment proteins

0.0 0 01 μm μm 0.01

Figure 4.27 A cadherin-mediated junction. The cadherin molecule is anchored to actin in the cytoskeleton and passes through the membrane to interact with the cadherin of an adjoining cell. 86 part II Biology of the Cell

Hemidesmosomes and focal adhesions connect cells to the basal lamina or other ECM. In this case the proteins that interact with the ECM are called integrins. The integrins are members of a large superfamily of cell-surface receptors that bind to a protein component of the ECM. At least 20 different integrins exist, each with a differently shaped binding domain. These junctions also connect to the cytoskeleton of cells: actin filaments at focal adhesions and intermediate filaments at hemidesmosomes.

Septate, or tight, junctions Septate junctions are found in both invertebrates and vertebrates and form a barrier that can seal off a sheet of cells. The proteins found at these junctions have been given different names in different systems; in Drosophila, the proteins include Discs Large and Neurexin. Their wide distribution indicates that they probably evolved soon after or with adherens junctions. Tight junctions are unique to vertebrates and contain proteins called Claudins because of their ability to occlude or block substances from passing between cells. This form of junction between cells acts as a wall within the tissue, keeping molecules on one side or the other (figure 4.28a). Creating sheets of cells.   The cells that line an animal’s digestive tract are organized in a sheet only one cell thick. One surface of the sheet faces the inside of the tract, and the other faces the extracellular space, where blood vessels are located. Tight junctions encircle each cell in the sheet, like a belt cinched around a person’s waist. The junctions between neighboring cells are so securely attached that there is no space between them for leakage. Hence, nutrients absorbed from the food in the digestive tract must pass directly through the cells in the sheet to enter the bloodstream because they cannot pass through spaces between cells. The tight junctions between the cells lining the digestive tract also partition the plasma membranes of these cells into separate compartments. Transport proteins in the membrane facing the inside of the tract carry nutrients from that side to the cytoplasm of the cells. Other proteins, located in the membrane on the opposite side of the cells, transport those nutrients from the cytoplasm to the extracellular fluid, where they can enter the bloodstream. Tight junctions effectively segregate the proteins on opposite sides of the sheet, preventing them from drifting within the membrane from one side of the sheet to the other. When tight junctions are experimentally disrupted, just this sort of migration occurs.

Communicating junctions The proteins involved in the junctions previously described can be found in some single-celled organisms as well. The evolution of multicellularity also led to a new form of cellular connection: the communicating junctions. These junctions allow communication between cells by diffusion through small openings. Communicating junctions permit small molecules or ions to pass from one cell to the other. In animals, these direct communication channels between cells are called gap junctions, and in plants, plasmodesmata. Gap junctions in animals. Gap junctions are found in both invertebrates and vertebrates. In invertebrates they are formed by proteins known as pannexins. In vertebrates p­ annexin-base gap

Tight junction Adjacent plasma membranes Tight junction proteins Intercellular space

a.

0.2 μm

Microvilli

Adhesive junction (desmosome) Intercellular space Adjacent plasma membranes

Tight junction

Cadherin Cytoplasmic protein plaque Cytoskeletal filaments anchored to plaque

b.

Adhesive junction (desmosome)

Intermediate filament

0.1 μm Communicating junction

Communicating junction

Intercellular space Connexon Two adjacent connexons forming an open channel between cells Channel (diameter 1.5 nm) Adjacent plasma membranes

c.

Basal lamina

1.4 μm

Figure 4.28 Cell junction types in animal epithelium. Here, the diagram of gut epithelial cells on the right illustrates the comparative structures and locations of common cell junctions. The detailed models on the left show the structures of the three major types of cell junctions: (a) tight junction; (b) adhesive junction—the example shown is a desmosome; (c) communicating junction—the example shown is a gap junction.  Don W. Fawcett/Science Source

junctions exist, but there is an additional type based on similar proteins called connexons. In each case, a structure is formed by complexes of six identical transmembrane proteins (figure 4.28c). The proteins are arranged in a circle to create a channel through the plasma membrane that protrudes several nanometers from the cell surface. A gap junction forms when the connexons/pannexins of two cells align perfectly, creating an open channel that spans the plasma membranes of both cells. Gap junctions provide passageways large enough to permit small substances, such as simple sugars and amino acids, to pass from one cell to the next. Yet the passages are small enough to prevent the passage of larger molecules, such as proteins. Gap junction channels are dynamic structures that can open or close in response to a variety of factors, including Ca2+ and

H+ ions. This gating serves at least one important function. When a cell is damaged, its plasma membrane often becomes leaky. Ions in high concentrations outside the cell, such as Ca2+, flow into the damaged cell and close its gap junction channels. This isolates the cell and prevents the damage from spreading. Plasmodesmata in plants. In plants, cell walls separate every cell from all others. Cell–cell junctions occur only at holes or gaps in the walls, where the plasma membranes of adjacent cells can come into contact with one another. Cytoplasmic connections that form across the touching plasma ­ membranes are called plasmodesmata (singular, plasmodesma) (figure 4.29). The majority of living cells within a higher plant are connected to their neighbors by these junctions. chapter

4 Cell Structure 87

Primary cell wall

Middle lamella

Smooth ER

Plasma membrane

Plasmodesmata function much like gap junctions in animal cells, although their structure is more complex. Unlike gap junctions, plasmodesmata are lined with plasma membrane and contain a central tubule that connects the ER of the two cells.

Plasmodesma

Learning Outcomes Review 4.8

Central tubule Cell 1

Cell 2

Figure 4.29 Plasmodesmata. Plant cells can communicate

through specialized openings in their cell walls, called plasmodesmata, which connect the cytoplasm of adjoining cells.

The evolution of multicellularity required the acquisition of cell adhesion molecules to connect cells together. Cell connections fall into three basic categories: (1) adhesive junctions provide strength and flexibility; (2) tight, or septate, junctions help to make sheets of cells that form watertight seals; and (3) communicating junctions, including gap junctions in animals and plasmodesmata in plants, allow passage of some materials between cells. Cells in multicellular organisms have distinct identity and connections. Cell identity is conferred by surface glycoproteins, which include the MHC proteins that are important in the immune system. ■■

How do cell junctions help to form tissues?

Chapter Review Dr. Gopal Murti/Science Source

4.1 Cell Theory

Cell theory is the unifying foundation of cell biology. All organisms are composed of one or more cells. Cells arise only by division of preexisting cells.

Bacterial and archaeal flagellar structures evolved independently. Bacterial flagella and the archaeal archaellum are an example of convergent evolution. They both show similar rotation, but the motive force is different.

Cell size is limited. Cell size is constrained by the diffusion distance. As cell size increases, diffusion becomes inefficient.

4.3 Eukaryotic Cells (figures 4.6 and 4.7)

Microscopes allow visualization of cells and components. Magnification gives better resolution than is possible with the naked eye. Staining with chemicals enhances contrast of structures.

The nucleus acts as the information center. The nucleus is surrounded by an envelope of two phospholipid bilayers; the outer layer is contiguous with the ER. Pores allow exchange of small molecules. The nucleolus is a region of the nucleoplasm where rRNA is transcribed and ribosomes are assembled. In most prokaryotes, DNA is organized into a single circular chromosome. In eukaryotes, numerous chromosomes are present.

All cells share simple structural features. All cells have centrally located DNA, a semifluid cytoplasm, and an enclosing plasma membrane.

4.2 Prokaryotic Cells (figure 4.3) Prokaryotic cells are more complex than we thought. Some species have membrane-bound organelles, but none found in all cells. Many bacteria have compartments formed by semipermeable protein shells. Prokaryotes lack a cytoskeleton, but have proteins related to eukaryotic actin and tubulin. Bacterial cell walls consist of peptidoglycan. Peptidoglycan is composed of carbohydrate cross-linked with short peptides. Archaea have unusual membrane lipids. Archaeal cell walls do not contain peptidoglycan, and they have unique plasma membranes. 88 part II Biology of the Cell

Eukaryotic cells have a membrane-bounded nucleus, an endomembrane system, and many different organelles.

Ribosomes are the cell’s protein synthesis machinery. Ribosomes translate mRNA to produce polypeptides. They are found in all cell types.

4.4 The Endomembrane System (figures 4.10–4.15) The endoplasmic reticulum (ER) divides the cytoplasm into compartments, and creates channels and passages within the cell. The rough ER is a site of protein synthesis. The rough ER (RER) is studded with ribosomes that synthesize membrane proteins or proteins that will be exported. These may be modified in the RER.

The SER has multiple roles. The SER lacks ribosomes; it is involved in carbohydrate and lipid synthesis and detoxification. The SER also stores Ca2+. The Golgi apparatus sorts and packages proteins. The Golgi apparatus receives vesicles from the ER, modifies and packages macromolecules, and transports them. Lysosomes contain digestive enzymes. Lysosomes break down macromolecules and recycle the components of old organelles. Lysosomal enzymes are active at acid pH. Lipid droplets store neutral lipids. Lipid droplets consist of neutral lipids surrounded by a phospholipid monolayer. Microbodies are a diverse category of organelles. Peroxisomes contain enzymes to oxidize fats. Plants use vacuoles for storage and water balance. The membrane of the central vacuole has channels to transport water. Some fungi and protists also use vacuoles for water balance.

4.5 Mitochondria and Chloroplasts: Cellular

Generators

Mitochondria and chloroplasts have a double-membrane structure, contain their own DNA, and can divide independently. Mitochondria generate most cellular ATP. The inner membrane of mitochondria is extensively folded into layers called cristae. Proteins on the surface and in the inner membrane carry out metabolism to produce ATP (figure 4.16). Chloroplasts use light to generate ATP and sugars. Chloroplasts capture light energy via thylakoid membranes arranged in stacks called grana, and use it to synthesize glucose (figure 4.17). Mitochondria and chloroplasts arose by endosymbiosis. The endosymbiont theory proposes that mitochondria and chloroplasts were once prokaryotes engulfed by another cell.

4.6 The Cytoskeleton The cytoskeleton consists of crisscrossed protein fibers that support the shape of the cell and anchor organelles (figure 4.19).

Three types of fibers compose the cytoskeleton. Actin filaments, or microfilaments, are long, thin polymers involved in cellular movement. Microtubules are hollow structures that move materials within a cell. Intermediate filaments serve a wide variety of functions. Centrosomes are microtubule-organizing centers. Centrosomes help assemble the nuclear division apparatus of animal cells (figure 4.20). The cytoskeleton helps move materials within cells. Molecular motors move vesicles along microtubules, like a train on a railroad track. Kinesin and dynein are two motor proteins.

4.7 Extracellular Structures and Cell Movement Some cells crawl. Cell crawling occurs as actin polymerization forces the cell membrane forward, while myosin pulls the cell body forward. Flagella and cilia aid movement. Eukaryotic flagella have a 9 + 2 structure and arise from a basal body. Cilia are shorter and more numerous than flagella. Plant cell walls provide protection and support. Plants have cell walls composed of cellulose fibers. The middle lamella, between cell walls, holds adjacent cells together. Animal cells secrete ECM. Glycoproteins are the main component of the extracellular matrix (ECM) of animal cells.

4.8 Cell-to-Cell Interactions (figure 4.28) Surface proteins give cells identity. Glycolipids and MHC proteins on cell surfaces help distinguish self from nonself. Cell connections mediate cell-to-cell adhesion. Cell junctions include tight junctions, adhesive junctions, and communicating junctions. In animals, gap junctions allow the passage of small molecules between cells. In plants, plasmodesmata penetrate the cell wall and connect cells.

chapter

4 Cell Structure 89

Visual Summary Genetic material Cytoplasm

have have

All cells

Plasma membrane

have have

Ribosomes Cilia and flagella

Archaea Eukaryotic cells

Prokaryotic cells Bacteria

Nucleus

have

contain

have

have no

have

Extracellular structures

Animal extracellular matrix

have a

Organelles

Cytoskeleton 3 types

surrounded by including

Nuclear envelope

Cell walls function

Maintain shape

Protection

Flagella

Mitochondria

Chloroplast

allow

function

function

Movement

Cellular respiration

Actin filaments

Intermediate filaments

Microtubules

Endomembrane system

Photosynthesis

made of

Endoplasmic reticulum

Lysosomes

Golgi function Sort & package protein

Rough ER

Protein synthesis

function

Vesicles

Central vacuole

function

function

Digestion Transport

Smooth ER function

function

90 part II Biology of the Cell

Plant cell walls

include

Lipid synthesis

Calcium storage

Storage & water balance

Review Questions Dr. Gopal Murti/Science Source

U N D E R S TA N D 1. Which of the following statements is NOT part of the cell theory? a. b. c. d.

All organisms are composed of one or more cells. Cells come from other cells by division. Cells are the smallest living things. Eukaryotic cells have evolved from prokaryotic cells.

2. All cells have all of the following except a. b.

plasma membrane. genetic material.

c. d.

cytoplasm. cell wall.

3. Eukaryotic cells are more complex than prokaryotic cells. Which of the following are found only in a eukaryotic cell? a. b.

Cell wall Plasma membrane

c. d.

Endoplasmic reticulum Ribosomes

4. Which of the following are differences between bacteria and archaea? a. b. c. d.

The molecular architecture of their cell walls The type of ribosomes found in each Archaea have an internal membrane system that bacteria lack. Both a and b are correct.

5. The cytoskeleton includes a. b. c. d.

microtubules made of actin filaments. microfilaments made of tubulin. intermediate filaments made of twisted fibers of vimentin and keratin. SER.

6. The SER is a. b. c. d.

involved in protein synthesis. a site of protein glycosylation. used to store a variety of ions. the site of lipid and membrane synthesis.

7. Plasmodesmata in plants and gap junctions in animals are functionally similar in that a. b. c. d.

each is used to anchor layers of cells. they form channels between cells that allow diffusion of small molecules. they form tight junctions between cells. they are anchored to the extracellular matrix.

A P P LY 1. The most important factor that limits the size of a cell is the a. b. c. d.

quantity of proteins and organelles a cell can make. rate of diffusion of small molecules. surface area-to-volume ratio of the cell. amount of DNA in the cell.

2. All eukaryotic cells possess each of the following except a. b.

mitochondria. cell wall.

c. d.

cytoskeleton. nucleus.

3. Adherens junctions, which contain cadherin, are found in all animals. Given this, which of the following predictions is most likely? a. b.

Cadherins would not be found in the ancestor to all animals. Cadherins would be found in prokaryotes.

c. d.

Cadherins would be found in the ancestor to all animals. Cadherins would be found in vertebrates but not invertebrates.

4. Different motor proteins like kinesin and myosin are similar in that they can a. b. c. d.

interact with microtubules. use energy from ATP to produce movement. interact with actin. do both a and b.

5. The protein sorting pathway involves the following organelles/ compartments in order: a. b. c. d.

SER, RER, transport vesicle, Golgi. RER, lysosome, Golgi. RER, transport vesicle, Golgi, final destination. Golgi, transport vesicle, RER, final destination.

6. Chloroplasts and mitochondria have many common features because both a. b. c. d.

are present in plant cells. arose by endosymbiosis. function to oxidize glucose. function to produce glucose.

7. Eukaryotic cells are composed of three types of cytoskeletal filaments. How are these three filaments similar? a. b. c. d.

They contribute to the shape of the cell. They are all made of the same type of protein. They are all the same size and shape. They are all equally dynamic and flexible.

SYNTHESIZE 1. The SER is the site of synthesis of the phospholipids that make up all the membranes of a cell—especially the plasma membrane. Use the diagram of an animal cell (figure 4.6) to trace a pathway that would carry a phospholipid molecule from the SER to the plasma membrane. What endomembrane compartments would the phospholipids travel through? How can a phospholipid molecule move between membrane compartments? 2. Use the information provided in table 4.3 to develop a set of predictions about the properties of mitochondria and chloroplasts if these organelles were once free-living prokaryotic cells. How do your predictions match with the evidence for endosymbiosis? 3. Eukaryotes are thought to be more closely related to archaea than bacteria. Does the nature of membrane lipids in the three groups argue for or against this? How would you rationalize the differences in membrane lipids in the three groups? 4. The protist Giardia intestinalis is the organism associated with water-borne diarrheal diseases. Giardia is an unusual eukaryote because it seems to lack mitochondria. Provide two possible evolutionary scenarios for this in the context of the endosymbiotic theory.

chapter

4 Cell Structure 91

5

CHAPTER

Membranes Chapter Contents 5.1

The Structure of Membranes

5.2

Phospholipids: The Membrane’s Foundation

5.3

Proteins: Multifunctional Components

5.4

Passive Transport Across Membranes

5.5

Active Transport Across Membranes

5.6

Bulk Transport by Endocytosis and Exocytosis

100 nm Keith R. Porter/Science Source

100 nm

Visual Outline Fluid mosaic model

Cellular membranes

mediate

Introduction

Transport 3 types

Consists of Passive transport Phospholipid bilayer

3 types

moves moves down up

Bulk transport

Concentration gradient

Transmembrane proteins

Simple diffusion

Cytoskeleton

Osmosis

Cell surface markers

Facilitated diffusion

Endocytosis: Dr. Edwin P. Ewing, Jr./CDC

Active transport

types Requires energy

Endocytosis Exocytosis

ATP

A cell’s interactions with the environment are critical, a give-and-take that never ceases. Without it, life could not exist. Living cells are encased within a lipid membrane through which few water-soluble substances can pass. This plasma membrane also contains protein passageways that permit specific substances to move into and out of the cell and allow the cell to exchange information with its environment. Eukaryotic cells also contain internal membranes like those of the mitochondrion and endoplasmic reticulum pictured here. We call the delicate skin of lipids with embedded protein molecules that encase the cell a plasma membrane. This chapter examines the structure and function of cellular membranes.

5.1 The Structure of Membranes Learning Outcomes 1. Describe the components of biological membranes. 2. Explain the fluid mosaic model of membrane structure.

The membranes that encase all living cells are two phospholipid sheets that are only 5 to 10 nm thick; more than 10,000 of these sheets piled on one another would just equal the thickness of this sheet of paper. Biologists established the components of membranes—not only lipids, but also proteins and other molecules—through biochemical assays, but the organization of the membrane components remained elusive. We begin by considering the theories that have been advanced about the structure of the plasma membrane. We then look at the individual components of membranes more closely.

The fluid mosaic model shows proteins embedded in a fluid lipid bilayer The lipid layer that forms the foundation of a cell’s membranes is a bilayer formed of phospholipids. These phospholipids include

Polar Hydrophilic Heads

Cellular membranes consist of four component groups A eukaryotic cell contains many membranes. Although they are not all identical, they share the same fundamental architecture. Cell membranes are assembled from four components (table 5.1):

Figure 5.1 Different views of phospholipid structure. Phospholipids are

N+(CH3)3

CH2

Nonpolar Hydrophobic Tails

primarily the glycerol phospholipids (figure 5.1), and the sphingolipids such as sphingomyelin (figure 5.2). Note that although these look superficially similar, they are built on a different carbon skeleton. For many years, biologists thought that the protein components of the cell membrane covered the inner and outer surfaces of the phospholipid bilayer like a coat of paint. An early model portrayed the plasma membrane as a sandwich: a phospholipid bilayer between two layers of globular protein. In 1972, S. Jonathan Singer and Garth J. Nicolson revised the model in a simple but profound way: they proposed that the globular proteins are inserted into the lipid bilayer, with their nonpolar segments in contact with the nonpolar interior of the bilayer and their polar portions protruding out from the membrane surface. In this model, called the fluid mosaic model, a mosaic of proteins floats in or on the fluid lipid bilayer like boats on a pond (figure 5.3). We now recognize two categories of membrane proteins based on their association with the membrane. Integral ­membrane proteins are embedded in the membrane, and p ­ eripheral proteins are associated with the surface of the membrane.

CH2

composed of glycerol (pink) linked to two fatty acids and a phosphate group. The phosphate group ( yellow) can have additional molecules attached, such as the positively charged choline ( green) shown. Phosphatidylcholine is a common component of membranes. It is shown in (a) with its chemical formula, (b) as a space-filling model, and (c) as the icon that is used in most of the figures in this chapter.

O O

P

O−

O H H2C

C

O

O

C O CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C O CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

a. Formula

CH2

b. Space-filling model

c. Icon chapter

5 Membranes 93

Polar Hydrophilic Heads

O −O

H

Nonpolar Hydrophobic Tails

HO

P

CH3 O

N+

O

CH3

CH2 H

O

C

N

C

C

H

CH2

CH

CH2

CH

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

CH3

CH2 CH3

a. Formula

b. Space-filling model

Figure 5.2 Sphingomyelin. Sphingomyelin is a sphingolipid found in animal cells. a. Formula. b. Space-filling model. 

(b): Whitney L. Stutts, University of Florida

1. Phospholipid bilayer. Every cell membrane is composed of phospholipids in a bilayer. The other components of the membrane are embedded within the bilayer, which provides a flexible matrix and, at the same time, imposes a barrier to permeability. Animal cell membranes also contain cholesterol, a steroid with a polar hydroxyl group (–OH). Plant cells have other sterols, but little or no cholesterol. 2. Transmembrane proteins. A major component of every membrane is a collection of proteins that float in the lipid bilayer. These proteins have a variety of functions, including transport and communication across the membrane. Many integral membrane proteins are not fixed in position. They can move about, just as the phospholipid molecules do. Some membranes are crowded with proteins, but in others, the proteins are more sparsely distributed. 3. Interior protein network. Membranes are structurally supported by intracellular proteins that reinforce the membrane’s shape. For example, a red blood cell has a characteristic biconcave shape because a scaffold made of a protein called spectrin links proteins in the plasma membrane with actin filaments in the cell’s cytoskeleton. Membranes use networks of other proteins to control the lateral movements of some key membrane proteins, anchoring them to specific sites. 4. Cell-surface markers. As you learned in chapter 4, membrane sections assemble in the endoplasmic reticulum (ER), transfer to the Golgi apparatus, and then are transported to the plasma membrane. The ER adds chains of sugar molecules to membrane proteins and lipids, converting them into glycoproteins and glycolipids. Different cell types exhibit different varieties of these glycoproteins and glycolipids on their surfaces, which act as cell identity markers.

Figure 5.3 The fluid mosaic model of cell membranes. Integral

proteins protrude through the plasma membrane, with nonpolar regions that tether them to the membrane’s hydrophobic interior. Carbohydrate chains are often bound to the extracellular portion of these proteins, forming glycoproteins. Peripheral membrane proteins are associated with the surface of the membrane. Membrane phospholipids can be modified by the addition of carbohydrates to form glycolipids. Inside the cell, actin filaments and intermediate filaments interact with membrane proteins. Outside the cell, many animal cells have an elaborate extracellular matrix composed primarily of glycoproteins.

Extracellular matrix protein

Glycoprotein Glycolipid Integral proteins

Glycoprotein

Actin filaments of cytoskeleton

Cholesterol

Peripheral protein Intermediate filaments of cytoskeleton

94 part II Biology of the Cell

TA B L E 5 .1 Component

Components of the Cell Membrane Composition

Function

How It Works

Example

Phospholipid bilayer

Phospholipid molecules

Provides permeability barrier, matrix for proteins

Excludes water-soluble molecules from nonpolar interior of bilayer and cell

Bilayer of cell is impermeable to large water-soluble molecules, such as glucose

Transmembrane proteins

Carriers

Actively or passively transport molecules across membrane

Move specific molecules through the membrane in a series of conformational changes

Glycophorin carrier for sugar transport; sodium–potassium pump

Channels

Passively transport molecules across membrane

Create a selective tunnel that acts as a passage through membrane

Sodium and potassium channels in nerve, heart, and muscle cells

Receptors

Transmit information into cell

Signal molecules bind to cell-surface portion of the receptor protein. This alters the portion of the receptor protein within the cell, inducing activity

Specific receptors bind peptide hormones and neurotransmitters

Spectrins

Determine shape of cell

Form supporting scaffold beneath membrane, anchored to both membrane and cytoskeleton

Red blood cell

Clathrins

Anchor certain proteins to specific sites, especially on the exterior plasma membrane in receptor-mediated endocytosis

Proteins line coated pits and facilitate binding to specific molecules

Localization of low-density lipoprotein receptor within coated pits

Glycoproteins

“Self” recognition

Create a protein/carbohydrate chain shape characteristic of individual

Major histocompatibility complex protein recognized by immune system

Glycolipid

Tissue recognition

Create a lipid/carbohydrate chain shape characteristic of tissue

A, B, O blood group markers

Interior protein network

Cell-surface markers

Cellular membranes have an organized substructure Originally, it was believed that because of its fluidity, the plasma membrane was uniform, with lipids and proteins free to diffuse rapidly in the plane of the membrane. However, in the last decade evidence has accumulated suggesting the plasma membrane is not homogeneous and contains microdomains with distinct lipid and protein composition. This was first observed in epithelial cells in which the lipid composition of the apical and basal membranes was shown to be distinctly different. Theoretical work also showed that lipids can exist in either a disordered or an ordered phase within a bilayer. This led to the idea of lipid microdomains called lipid rafts that are heavily enriched in cholesterol and sphingolipids. These lipids appear to interact with each other, and with raft-associated proteins—together forming an ordered structure. These structures are now technically defined as “dynamic nanometer-sized, sterol and sphingolipid-enriched protein assemblies.” There is evidence that signaling molecules, such as the B- and T-cell receptors discussed in chapter 50, associate with lipid rafts and that this association affects their function.

Electron microscopy has provided structural evidence Electron microscopy allows biologists to examine the delicate, filmy structure of a cell membrane. We discussed two types of electron microscopes in chapter 4: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). Both provide illuminating views of membrane structure. Specimens are prepared for examination in a TEM by embedding the tissue in a hard epoxy matrix. This epoxy block is

then cut into many sections with a microtome, a machine with a very sharp blade that makes incredibly thin, transparent “epoxy shavings” less than 1 µm thick. These shavings are placed on a grid, and a beam of electrons is directed through the grid with the TEM. At the high magnification an electron microscope provides, resolution is good enough to reveal the double layers of a membrane. False color can be added to the micrograph to enhance detail (figure 5.4). Freeze-fracturing a specimen is another way to visualize the inside of the membrane (figure 5.5). The tissue is embedded in a medium and quick frozen with liquid nitrogen. The frozen tissue is then “tapped” with a knife, causing a crack between the phospholipid layers

Cell 1

Plasma membrane of cell 1

Plasma membrane of cell 2 Cell 2

25 nm

Figure 5.4 Electron micrograph of adjacent cell membranes. The micrograph shows two adjacent cells. The

structure of the plasma membrane of each cell is clear, and the micrograph has been false colored to emphasize the bilayer nature of the membranes.  Don W. Fawcett/Science Source chapter

5 Membranes 95

2. The cell often fractures through the interior, hydrophobic area of the lipid bilayer, splitting the plasma membrane into two layers.

1. A cell frozen in medium is cracked with a knife blade.

Medium

3. The plasma membrane separates such that proteins and other embedded membrane structures remain within one or the other layers of the membrane.

4. The exposed membrane is coated with platinum, which forms a replica of the membrane. The underlying membrane is dissolved away, and the replica is then viewed with electron microscopy.

Fractured upper half of lipid bilayer Exposed lower half of lipid bilayer

Cell Knife

Figure 5.5 Viewing a plasma membrane with freeze-fracture microscopy. (4): Don W. Fawcett/Science Source

of membranes. Proteins, carbohydrates, pits, pores, channels, or any other structure affiliated with the membrane will pull apart (whole, usually) and stick with one or the other side of the split membrane. Next, a very thin coating of platinum is evaporated onto the fractured surface, forming a replica or “cast” of the surface. After the topography of the membrane has been preserved in the cast, the actual tissue is dissolved away, and the cast is examined with electron microscopy, creating a textured and three-dimensional view of the membrane.

Learning Outcomes Review 5.1

Cellular membranes contain four components: (1) a phospholipid bilayer, (2) transmembrane proteins, (3) an internal protein network providing structural support, and (4) cell-surface markers composed of glycoproteins and glycolipids. The fluid mosaic model of membrane structure includes both the fluid nature of the membrane and the mosaic composition of proteins floating in the phospholipid bilayer. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have provided evidence supporting the fluid mosaic model. ■■

If the plasma membrane were just a phospholipid bilayer, how would this affect its function?

5.2 Phospholipids:

The Membrane’s Foundation

Learning Outcomes 1. List the different components of phospholipids. 2. Explain how membranes form spontaneously. 3. Describe the factors involved in membrane fluidity. 96 part II Biology of the Cell

Exposed lower half of lipid bilayer

0.15 μm External surface of plasma membrane

Lipidomics, the field defining the number and biological function of lipids, is revealing a significant diversity in membrane lipids. Although there are over 1000 distinct lipids identified in cells, we can organize them into only three classes: glycerol phospholipids (see figure 5.1), sphingolipids (see figure 5.2), and sterols such as cholesterol. The classical phospholipid bilayer consists of a combination of glycerol phospholipids and sphingolipids. The glycerol phospholipids are the most diverse, with head groups that can have both positive and negative charge (zwitterionic), or primarily negative charge (anionic). Phospholipids can also vary in the length and composition of the fatty acid tail, with it being either saturated or cis-unsaturated (see section 3.5). Sphingolipids usually contain saturated hydrocarbon chains. These components are not uniformly distributed in biological membranes, and different cellular compartments have distinct membrane lipid composition, as discussed later in this section.

Phospholipids spontaneously form bilayers Phospholipids spontaneously form bilayers because of their amphipathic structure. The polar head groups are hydrophilic, whereas the nonpolar hydrocarbon tails are hydrophobic. The two nonpolar fatty acids extend in one direction, roughly parallel to each other, and the polar phosphate group points in the other direction. To represent this structure, phospholipids are often diagrammed as a polar head with two dangling nonpolar tails, as in figure 5.1c. What happens when a collection of phospholipid molecules is placed in water? The polar water molecules repel the long, nonpolar tails of the phospholipids while seeking partners for hydrogen bonding. Because of the polar nature of the water molecules, the nonpolar tails of the phospholipids end up packed closely together, sequestered as far as possible from water. Every phospholipid molecule is oriented with its polar head toward water and its nonpolar tails away. When two layers form with the tails facing each other, no tails ever come in contact with water. The resulting structure is the phospholipid bilayer. Phospholipid bilayers form spontaneously, driven by the tendency of water molecules to form the maximum number of hydrogen bonds.

SCIENTIFIC THINKING Hypothesis: The plasma membrane is fluid, not rigid. Prediction: If the membrane is fluid, membrane proteins may diffuse laterally. Test: Fuse mouse and human cells, then observe the distribution of membrane proteins over time by labeling specific mouse and human proteins. Human cell

Mouse cell

Fuse cells

Intermixed membrane proteins

Allow time for mixing to occur Result: Over time, hybrid cells show increasingly intermixed proteins. Conclusion: At least some membrane proteins can diffuse laterally in the membrane. Further Experiments: Can you think of any other explanation for these observations? What if newly synthesized proteins were inserted into the membrane during the experiment? How could you use this basic experimental design to rule out this or other possible explanations?

mono cis-unsaturated, tend to make the membrane less fluid, as they pack well. Similarly, the sphingolipids, which are usually saturated, also make the membrane less fluid. Changes in the environment can have drastic effects on the plasma membrane of single-celled organisms such as bacteria. Increasing temperature makes a membrane more fluid, and decreasing temperature makes it less fluid. Bacteria have evolved mechanisms to maintain a constant membrane fluidity despite fluctuating temperatures. Some bacteria contain enzymes called fatty acid desaturases that can introduce double bonds into fatty acids in membranes. Genetic studies, involving either the inactivation of these enzymes or the introduction of them into cells that normally lack them, indicate that the action of these enzymes confers cold tolerance. At colder temperatures, the double bonds introduced by fatty acid desaturase make the membrane more fluid, counteracting the environmental effect of reduced temperature.

Phospholipid composition affects membrane structure Although most lipids are synthesized in the ER, the composition of the ER membrane, the Golgi stack, and the plasma membrane are quite distinct. These differences in lipid composition affect the structure and function of each membrane compartment (figure 5.7).

Endoplasmic Reticulum • Major packing defects • Thin –

Figure 5.6 Test of membrane fluidity.

– 20 aa

The nonpolar interior of a lipid bilayer impedes the passage of any water-soluble substances through the bilayer, just as a layer of oil impedes the passage of a drop of water. This barrier to water-soluble substances is the key biological property of the lipid bilayer.

Unsaturated

TMD Cytosol

The phospholipid bilayer is fluid A lipid bilayer is stable because water’s affinity for hydrogen bonding never stops. Just as surface tension holds a soap bubble together, even though it is made of a liquid, so the hydrogen bonding of water holds a membrane together. Although water drives phospholipids into a bilayer configuration, it does not have any effect on the mobility of phospholipids and their nonlipid neighbors in the bilayer. Because phospholipids interact relatively weakly with one another, individual phospholipids and unanchored proteins are comparatively free to move about within the membrane. This can be demonstrated vividly by fusing cells and watching their proteins intermix with time (figure 5.6).

Membrane fluidity varies with lipid composition The degree of membrane fluidity changes with the composition of the membrane itself. Much as triglycerides can be solid or liquid at room temperature, depending on their fatty acid composition, membrane fluidity can be altered by changing the membrane’s lipid composition. Glycerol phospholipids that are saturated, or

Plasma Membrane Saturated

Cholesterol

25 aa –

–

–

• Minor packing defects • Thick

–

–

–

Anionic head group

–

–

Cytosol

Figure 5.7 Comparing endoplasmic reticulum and plasma membrane. The different composition of membrane lipids in the ER and the plasma membrane result in different membrane structures. The plasma membrane is thicker and less permeable; the ER membrane forms tubes and sheets and is more dynamic. chapter

5 Membranes 97

The plasma membrane has a high concentration of the cylinder-shaped phosphatidylcholine and sphingomyelin, which makes a dense gel-like membrane. This is given fluidity by the incorporation of cholesterol, which interacts with the nonpolar tails. Cholesterol is also concentrated in the outer leaflet compared to the cytoplasmic leaflet. The unsaturated hydrocarbon chains also make a thicker membrane, with the length of transmembrane domains for membrane proteins around 25 amino acids. The overall structure of the plasma membrane is a fluid, but relatively rigid, membrane that forms an excellent barrier. The ER membrane contains mainly unsaturated phospholipids that make the membrane more fluid, but also introduce curvature because they tend to form cones or inverted cones rather than cylinders. There is little to no cholesterol in the ER membrane. The ER membrane is also thinner than the plasma membrane, which is reflected in the length of around 20 amino acids for the transmembrane domains of ER proteins. The distinct lipid composition in different membranes is maintained by the cell using a combination of vesicle traffic, and proteins that transfer lipids from one membrane to another. These lipid transfer proteins hold lipids like a glove, shuttling them between membranes.

Learning Outcomes Review 5.2

Biological membranes contain glycerol phospholipids, sphingolipids, and cholesterol. In water, phospholipid molecules spontaneously form a bilayer, with phosphate groups facing out toward the water and lipid tails facing in, where they are sequestered from water. Membrane fluidity varies with composition and conditions: unsaturated fats disturb packing of the lipid tails and make the membrane more fluid, as do higher temperatures. The phospholipid composition of different membrane compartments varies despite lipid movement between compartments. ■■

Would a phospholipid bilayer form in a nonpolar solvent?

5.3 Proteins:

Multifunctional Components

Learning Outcomes 1. Illustrate the functions of membrane proteins. 2. Illustrate how proteins can associate with the membrane. 3. Identify a transmembrane domain.

Cell membranes contain a complex assembly of proteins enmeshed in the fluid soup of phospholipid molecules. This very flexible organization permits a broad range of interactions with the environment, some directly involving membrane proteins. 98 part II Biology of the Cell

Proteins and protein complexes perform key functions Although cells interact with their environment through their plasma membranes in many ways, we will focus on six key classes of mem­ brane protein in this chapter and in chapter 9 (figure 5.8): 1. Transporters. Membranes are very selective, allowing only certain solutes to enter or leave the cell, through either channels or carriers composed of proteins. 2. Enzymes. Cells carry out many chemical reactions on the interior surface of the plasma membrane, using enzymes attached to the membrane. 3. Cell-surface receptors. Membranes are exquisitely sensitive to chemical messages, which are detected by receptor proteins on their surfaces. 4. Cell-surface identity markers. Membranes carry cell-surface markers that identify them to other cells. Most cell types carry their own ID tags, specific combinations of cell-surface proteins and protein complexes such as glycoproteins that are characteristic of that cell type. 5. Cell-to-cell adhesion proteins. Cells use specific proteins to glue themselves to one another. Some act by forming temporary interactions, and others form a more permanent bond. (See chapter 4.) 6. Attachments to the cytoskeleton. Surface proteins that interact with other cells are often anchored to the cytoskeleton by linking proteins. 7. Proteins that affect membrane structure. Membranes tend to form spheres or sheets in aqueous solutions. Wedge-shaped proteins can cause membranes to bend, allowing the formation of tubes and folded sheets.

Structural features of membrane proteins relate to function As we’ve just detailed, membrane proteins can serve a variety of functions. These diverse functions arise from the diverse structures of these proteins, yet they also have common structural features related to their role as membrane proteins.

The anchoring of proteins in the bilayer Some membrane proteins are attached to the surface of the membrane by special molecules that associate strongly with phospholipids. Like a ship tied to a floating dock, these anchored proteins are free to move about on the surface of the membrane tethered to a phospholipid. The anchoring molecules are modified lipids that have (1) nonpolar regions that insert into the internal portion of the lipid bilayer and (2) chemical bonding domains that link directly to proteins.

?

Inquiry question According to the fluid mosaic model,

membranes are held together by hydrophobic interactions. Considering the forces that some cells may experience, why do membranes not break apart every time an animal moves?

Outside cell

Inside cell

Transporter

Enzyme

Cell-surface receptor

Cell-surface identity marker

Cell-to-cell adhesion

Attachment to the cytoskeleton

Figure 5.8 Functions of plasma membrane proteins. Membrane proteins act as transporters, enzymes, cell-surface receptors, and cell-surface identity markers, as well as aiding in cell-to-cell adhesion and securing the cytoskeleton. In contrast, other proteins actually span the lipid bilayer (transmembrane proteins). The part of the protein that extends through the lipid bilayer and that is in contact with the nonpolar interior are α helices or β-pleated sheets (see chapter 3) that consist of nonpolar amino acids. Because water avoids nonpolar amino acids, these portions of the protein are held within the interior of the lipid bilayer. The polar ends protrude from both sides of the membrane. Any movement of the protein out of the membrane, in either direction, brings the nonpolar regions of the protein into contact with water, which “shoves” the protein back into the interior. These forces prevent the transmembrane proteins from simply popping out of the membrane and floating away.

Transmembrane domains Cell membranes contain a variety of different transmembrane proteins, which differ in the way they traverse the lipid bilayer. The primary difference lies in the number of times that the protein crosses the membrane. Each membrane-spanning region is called a ­transmembrane domain. These domains are composed of hydrophobic amino acids usually arranged into α helices (figure 5.9). Proteins need only a single transmembrane domain to be anchored in the membrane, but they often have more than one such domain. An example of a protein with a single transmembrane domain is the linking protein that attaches the spectrin network of the cytoskeleton to the interior of the plasma membrane. Biologists classify some types of receptors based on the num­ber of transmembrane domains they have, such as G protein–coupled receptors with seven membrane-spanning domains (chapter 9). These receptors respond to external molecules, such as epinephrine, and initiate a cascade of events inside the cell.

a.

b.

Figure 5.9 Transmembrane domains. Integral membrane proteins have at least one hydrophobic transmembrane domain (shown in blue) to anchor them in the membrane. a. Receptor protein with seven transmembrane domains. b. Protein with single transmembrane domain.

Another example is bacteriorhodopsin, one of the key transmembrane proteins that carries out photosynthesis in halophilic (salt-loving) archaea. It contains seven nonpolar h­ elical segments that traverse the membrane, forming a structure within the membrane through which protons pass during the light-driven pumping of protons. chapter

5 Membranes 99

Pores Some transmembrane proteins have extensive nonpolar regions with secondary configurations of β-pleated sheets instead of α helices (see chapter 3). The β sheets form a characteristic motif, folding back and forth in a cylinder so the sheets arrange themselves like a pipe through the membrane. This forms a polar environment in the interior of the β sheets spanning the membrane. This socalled β barrel, open on both ends, is a common feature of the porin class of proteins that are found within the outer membrane of some bacteria. The openings allow molecules to pass through the membrane.

5.4  Passive

Transport Across Membranes

Learning Outcomes 1. Compare simple diffusion and facilitated diffusion. 2. Differentiate between channel proteins and carrier proteins. 3. Predict the direction of water movement by osmosis.

Bending membranes Many intracellular membranes have elaborate structure, like the folds and tubular networks of the ER. As discussed earlier, phospholipid composition can affect membrane shape, but proteins also affect membrane structure in several ways. Wedge-shaped transmembrane proteins called reticulons will cause a membrane to bend, or if there are enough, form a tube. These proteins are concentrated in tubular regions, and where membrane folds occur. Flattened regions can be stabilized by proteins that coat the membrane, preventing it from bending, and by adhesion proteins that hold adjacent folds together.

Learning Outcomes Review 5.3

Proteins in the membrane confer the main differences between membranes of different cells. Their functions include transport, enzymatic action, reception of extracellular signals, cell-to-cell interactions, and cell identity markers. Peripheral proteins can be anchored in the membrane by modified lipids. Integral membrane proteins span the membrane and have one or more hydrophobic regions, called transmembrane domains, that anchor them. ■■

?

a. 100 part II Biology of the Cell

Why are transmembrane domains hydrophobic?

Inquiry question Based only on

amino acid sequence, how would you recognize an integral membrane protein?

b.

c.

Many substances can move in and out of the cell without the cell’s having to expend energy. This type of movement is termed passive transport. Some ions and molecules can pass through the membrane fairly easily and do so because of a concentration gradient— a difference in concentration inside the membrane versus outside. Some substances also move in response to a concentration gradient, but do so through specific protein channels in the membrane.

Transport can occur by simple diffusion Molecules and ions dissolved in water are in constant random motion. This random motion causes a net movement of these substances from regions of high concentration to regions of lower concentration, a process called diffusion (figure 5.10). Net movement by diffusion will continue until the concentration is the same in all regions. Consider what happens when you add a drop of colored ink to a bowl of water. Over time diffusion of the ink molecules causes them to become dispersed throughout the solution. In the context of cells, we need to consider both the relative concentrations of a substance inside and outside the cell, and how easily the substance can cross the membrane. The major barrier to crossing a biological membrane is the hydrophobic interior that repels polar molecules but not nonpolar molecules. If a concentration difference exists for a nonpolar molecule, it will move across the membrane until the concentration is equal on both sides. At this point, movement in both directions still occurs, but there is no net change in either direction. This includes molecules like O2 and nonpolar organic molecules such as steroid hormones. The plasma membrane has limited permeability to small polar molecules and very limited permeability to larger polar molecules and ions. The movement of water, one of the most important polar molecules, is discussed separately.

d.

Figure 5.10 Diffusion. If a drop of colored ink is dropped into a beaker of water (a) its molecules dissolve (b) and diffuse (c). Eventually, diffusion results in an even distribution of ink molecules throughout the water (d).

Proteins allow membrane diffusion to be selective Many important molecules required by cells cannot easily cross the plasma membrane. These molecules can still enter the cell by diffusion through specific channel proteins or carrier proteins embedded in the plasma membrane, provided there is a higher concentration of the molecule outside the cell than inside. We call this process of diffusion mediated by a membrane protein facilitated diffusion. Channel proteins have a hydrophilic interior that provides an aqueous channel through which polar molecules can pass when the channel is open. Carrier proteins, in contrast to channels, bind specifically to the molecule they assist, much as an enzyme binds to its substrate. These channels and carriers are usually selective for one type of molecule, and thus the cell membrane is said to be selectively permeable.

Facilitated diffusion of ions through channels You saw in chapter 2 that atoms with an unequal number of protons and electrons have an electric charge and are called ions. Those that carry a positive charge are called cations and those that carry a negative charge are called anions. Because of their charge, ions interact well with polar molecules such as water, but are repelled by nonpolar molecules such as the interior of the plasma membrane. Therefore, ions cannot move between the cytoplasm of a cell and the extracellular fluid without the assistance of membrane transport proteins. Ion channels possess a hydrated interior that spans the membrane. Ions can diffuse through the channel in either direction, depending on their relative concentration across the membrane (figure 5.11). Some channel proteins can be opened or closed in response to a stimulus. These channels are called gated channels, and depending on the nature of the channel, the stimulus can be either chemical or electrical. Three conditions determine the direction of net movement of the ions: (1) their relative concentrations on either side of the membrane, (2) the voltage difference across the membrane and for the Extracellular fluid

Cytoplasm

a.

gated channels, and (3) the state of the gate (open or closed). A voltage difference is an electrical potential difference across the membrane called a membrane potential. Changes in membrane potential form the basis for transmission of signals in the nervous system and some other tissues. (We discuss this topic in detail in chapter 42.) Each type of channel is specific for a particular ion, such as calcium (Ca2+), sodium (Na+), potassium (K+), or chloride (Cl–), or in some cases, for more than one cation or anion. Ion channels play an essential role in signaling by the nervous system.

Facilitated diffusion by carrier proteins Carrier proteins can help transport both ions and other solutes, such as some sugars and amino acids, across the membrane. Transport through a carrier is still a form of diffusion and therefore requires a concentration difference across the membrane. Carriers must bind to the molecule they transport, so the relationship between concentration and rate of transport differs from that due to simple diffusion. As concentration increases, transport by simple diffusion shows a linear increase in rate of transport. But when a carrier protein is involved, a concentration increase means that more of the carriers are bound to the transported molecule. At high enough concentrations all carriers will be occupied, and the rate of transport will be constant. This means that the carrier exhibits saturation. This situation is somewhat like that of a stadium (the cell) where a crowd must pass through turnstiles to enter. If there are unoccupied turnstiles, you can go right through, but when all are occupied, you must wait. When ticket holders are passing through the gates at maximum speed, the rate at which they enter cannot increase, no matter how many are waiting outside.

Facilitated diffusion in red blood cells Several examples of facilitated diffusion can be found in the plasma membrane of vertebrate red blood cells (RBCs). One RBC carrier protein, for example, transports a different molecule in each direction: chloride ion (Cl–) in one direction and bicarbonate ion (­ HCO3–)

Extracellular fluid

Extracellular fluid

Cytoplasm

Cytoplasm

b.

Figure 5.11 Facilitated diffusion. Diffusion can be facilitated by membrane proteins. a. The movement of ions through a channel is

shown. On the left the concentration is higher outside the cell, so the ions move into the cell. On the right the situation is reversed. In both cases, transport continues until the concentration is equal on both sides of the membrane. At this point, ions continue to cross the membrane in both directions, but there is no net movement in either direction. b. Carrier proteins bind specifically to the molecules they transport. In this case, the concentration is higher outside the cell, so molecules bind to the carrier on the outside. The carrier’s shape changes, allowing the molecule to cross the membrane. This is reversible, so net movement continues until the concentration is equal on both sides of the membrane. chapter

5 Membranes 101

in the opposite direction. As you will learn in chapter 47, this carrier is important in the uptake and release of carbon dioxide. Glucose is transported in RBCs by facilitated diffusion through a specific carrier protein. The chemistry of this is interesting, as RBCs keep the internal concentration low by phosphorylating glucose as it enters the cell. This has several consequences: glucose phosphate no longer binds the transporter and therefore cannot leave the cell, it is the form of glucose that enters glycolysis (see chapter 7), and it maintains a steep concentration gradient for unphosphorylated glucose, favoring its entry into the cell. The glucose transporter is a carrier protein that specifically binds to glucose. When glucose binds to the transporter, this alters the shape of the carrier, pulling glucose through the bilayer and releasing it inside the plasma membrane. Releasing glucose causes the transporter to revert to its original shape so it can bind another glucose molecule outside the cell.

Urea molecule

Water molecules

Semipermeable membrane

Osmosis is the movement of water across membranes The cytoplasm of a cell contains ions and molecules, such as sugars and amino acids, dissolved in water. The mixture of these substances and water is called an aqueous solution. Water is termed the solvent, and the substances dissolved in the water are solutes. Both water and solutes tend to diffuse from regions of high concentration to ones of low concentration; that is, they diffuse down their concentration gradients. When two regions are separated by a membrane, what happens depends on whether the solutes can pass freely through that membrane. Most solutes, including ions and sugars, are not lipid-soluble and, therefore, are unable to cross the lipid bilayer. The concentration gradient of these solutes can lead to the movement of water.

Osmosis Water molecules interact with dissolved solutes by forming hydration shells around the charged solute molecules. When a membrane separates two solutions with different concentrations of solutes, the concentrations of free water molecules on the two sides of the membrane also differ. The side with higher solute concentration has tied up more water molecules in hydration shells and thus has fewer free water molecules. As a consequence of this difference, free water molecules move down their concentration gradient, toward the higher solute concentration. This net diffusion of water across a membrane toward a higher solute concentration is called osmosis (figure 5.12). The concentration of all solutes in a solution determines the osmotic concentration of the solution. If two solutions have unequal osmotic concentrations, the solution with the higher concentration is hypertonic (Greek hyper, “more than”), and the solution with the lower concentration is h ­ ypotonic (Greek hypo, “less than”). When two solutions have the same osmotic concentration, the solutions are isotonic (Greek iso, “equal”). The terms hyperosmotic, hypoosmotic, and i­sosmotic are also used to describe these conditions. A cell in any environment can be thought of as a plasma membrane separating two solutions: the cytoplasm and the extracellular fluid. The direction and extent of any diffusion of water across the plasma membrane is determined by comparing the osmotic strength of these solutions. Put another way, water diffuses out of a cell in a hypertonic solution (that is, the cytoplasm of the 102 part II Biology of the Cell

Figure 5.12 Osmosis. Concentration differences in charged or

polar molecules that cannot cross a semipermeable membrane result in movement of water, which can cross the membrane. Water molecules form hydrogen bonds with charged or polar molecules, creating a hydration shell around them in solution. A higher concentration of polar molecules (urea) shown on the left side of the membrane leads to water molecules gathering around each urea molecule. These water molecules are no longer free to diffuse across the membrane. The polar solute has reduced the concentration of free water molecules, creating a gradient. This causes a net movement of water by diffusion from right to left in the U-tube, raising the level on the left and lowering the level on the right.

cell is hypotonic, compared with the extracellular fluid). This loss of water causes the cell to shrink until the osmotic concentrations of the cytoplasm and the extracellular fluid become equal.

Aquaporins: Water channels The transport of water across the membrane is complex. Studies on artificial membranes show that water, despite its polarity, can cross the membrane, but this flow is limited. Water flow in living cells is facilitated by aquaporins, which are specialized channels for water. A simple experiment demonstrates this. If an amphibian egg is placed in hypotonic spring water (the solute concentration in the cell is higher than that of the surrounding water), it does not swell. If aquaporin mRNA is then injected into the egg, the channel proteins are expressed and appear in the egg’s plasma membrane. Water can now diffuse into the egg, causing it to swell. More than 11 different kinds of aquaporins have been found in mammals. These fall into two general classes: those that are specific for only water, and those that allow other small hydrophilic molecules, such as glycerol or urea, to cross the membrane as well. This latter class explains how some membranes allow the easy passage of small hydrophilic substances. The human genetic disease hereditary (nephrogenic) diabetes insipidus (NDI) has been shown to be caused by a nonfunctional aquaporin protein. This disease causes the excretion of large

Human Red Blood Cells

Hypertonic Solution

Isotonic Solution

Hypotonic Solution

Shriveled cells

Normal cells

Cells swell and eventually burst

5 μm

5 μm

Plant Cells

5 μm

Cell body shrinks from cell wall

Flaccid cell

Normal turgid cell

Figure 5.13 How solutes create osmotic pressure. In a hypertonic solution, water moves out of the cell, causing the cell to shrivel. In an isotonic solution, water diffuses into and out of the cell at the same rate, with no change in cell size. In a hypotonic solution, water moves into the cell. Direction and amount of water movement is shown with blue arrows (top). As water enters the cell from a hypotonic solution, pressure is applied to the plasma membrane until the cell ruptures. Water enters the cell due to osmotic pressure from the higher solute concentration in the cell. Osmotic pressure is measured as the force needed to stop osmosis. The strong cell wall of plant cells can withstand the hydrostatic pressure to keep the cell from rupturing. This is not the case with animal cells.  (left, middle, right): David M. Phillips/Science Source volumes of dilute urine, illustrating the importance of aquaporins to our physiology.

Osmotic pressure What happens to a cell in a hypotonic solution? (That is, the cell’s cytoplasm is hypertonic relative to the extracellular fluid.) In this situation, water diffuses into the cell from the extracellular fluid, causing the cell to swell. The pressure of the cytoplasm pushing out against the cell membrane, or hydrostatic pressure, increases. The amount of water that enters the cell depends on the difference in solute concentration between the cell and the extracellular fluid. This is measured as osmotic pressure, defined as the force needed to stop osmotic flow. If the membrane is strong enough, the cell reaches an equilibrium, at which the osmotic pressure, which tends to drive water into the cell, is exactly counterbalanced by the hydrostatic

pressure, which tends to drive water back out of the cell. However, a plasma membrane by itself cannot withstand large internal pressures, and an isolated cell under such conditions would burst like an overinflated balloon (figure 5.13). Accordingly, it is important for animal cells, which only have plasma membranes, to maintain osmotic balance. In contrast, the cells of prokaryotes, fungi, plants, and many protists are surrounded by strong cell walls, which can withstand high internal pressures without bursting.

Maintaining osmotic balance Organisms have developed many strategies for solving the dilemma posed by being hypertonic to their environment and therefore having a steady influx of water by osmosis: Extrusion. Some single-celled eukaryotes, such as the protist Paramecium, use organelles called contractile vacuoles to remove water. Each vacuole collects water from various parts of the cytoplasm and transports it to the central part of the vacuole, near the cell surface. The vacuole possesses a small pore that opens to the outside of the cell. By contracting rhythmically, the vacuole pumps out (extrudes) through this pore the water that is continuously drawn into the cell by osmotic forces. Isosmotic Regulation. Some organisms that live in the ocean adjust their internal concentration of solutes to match that of the surrounding seawater. Because they are isosmotic with respect to their environment, no net flow of water occurs into or out of these cells. Many terrestrial animals solve the problem in a similar way, by circulating a fluid through their bodies that bathes cells in an isotonic solution. The blood in your body, for example, contains a high concentration of the protein albumin, which elevates the solute concentration of the blood to match that of your cells’ cytoplasm. Turgor. Most plant cells are hypertonic to their immediate environment, containing a high concentration of solutes in their central vacuoles. The resulting internal hydrostatic pressure, known as turgor pressure, presses the plasma membrane firmly against the interior of the cell wall, making the cell rigid. Most green plants depend on turgor pressure to maintain their shape, and thus they wilt when they lack sufficient water.

Learning Outcomes Review 5.4

Passive transport involves diffusion, which requires a concentration gradient. Hydrophobic molecules can diffuse directly through the membrane (simple diffusion). Polar molecules and ions can also diffuse through the membrane, but only with the aid of a channel or carrier protein (facilitated diffusion). Channel proteins assist by forming a hydrophilic passageway through the membrane, whereas carrier proteins bind to the molecule they assist. Water passes through the membrane and through aquaporins in response to solute concentration differences inside and outside the cell. This process is called osmosis. ■■

If you require intravenous (IV) medication in the hospital, what should the concentration of solutes in the IV solution be relative to your blood cells? chapter

5 Membranes 103

5.5  Active

Transport Across Membranes

Learning Outcomes 1. Differentiate between active transport and diffusion. 2. Describe the function of the Na +/K + pump. 3. Explain the energetics of coupled transport.

Diffusion, facilitated diffusion, and osmosis are passive transport processes that move materials down their concentration gradients, but cells can also actively move substances across a cell membrane up their concentration gradients. This process requires the expenditure of energy, typically from ATP, and is therefore called active transport.

Active transport uses energy to move materials against a concentration gradient Like facilitated diffusion, active transport involves highly selective protein carriers within the membrane that bind to the transported substance, which could be an ion or a simple m ­ olecule, such as a sugar, an amino acid, or a nucleotide. These carrier proteins are called uniporters if they transport a single type of molecule and symporters or antiporters if they transport two different molecules together. Symporters transport two molecules in the same direction, and antiporters transport two molecules in opposite directions. These terms can also be used to describe facilitated diffusion carriers. Active transport is one of the most important functions of any cell. It enables a cell to take up additional molecules of a substance that is already present in its cytoplasm in concentrations higher than in the extracellular fluid. Active transport also enables a cell to move substances out of its cytoplasm and into the extracellular fluid, despite higher external concentrations. The use of energy from ATP in active transport may be direct or indirect. Let’s first consider how ATP is used directly to move ions against their concentration gradients.

The sodium–potassium pump runs directly on ATP More than one-third of all of the energy expended by an animal cell that is not actively dividing is used in the active transport of sodium (Na+) and potassium (K+) ions. Most animal cells have a low internal concentration of Na+, relative to their surroundings, and a high internal concentration of K+. They maintain these concentration differences by actively pumping Na+ out of the cell and K+ in. The remarkable protein that transports these two ions across the cell membrane is known as the sodium–­potassium pump (Na+/K+ pump) (figure 5.14). This carrier protein uses the energy stored in ATP to move these two ions. In this case, the energy is used to change the conformation of the carrier protein, which 104 part II Biology of the Cell

changes its affinity for either Na+ ions or K+ ions. This is an excellent illustration of how subtle changes in the structure of a protein affect its function. Cells use the Na+/K+ pump to create a high concentration of Na+ outside the cell, and a high concentration of K+ inside the cell. Movement against a concentration gradient can be done only by active transport; passive transport by diffusion would equalize concentration across the membrane. These gradients of Na+ and K+ are important in the creation of an electrical potential across the membrane, critical to the function of nerve cells (see chapter 42). The Na+/K+ pump works through the following series of conformational changes in the transmembrane protein (summarized in figure 5.14): Step 1. With ATP bound to the pump, three Na+ bind to the cytoplasmic side of the protein. Step 2. The bound ATP is hydrolyzed to ADP, transferring a phosphate to the pump. The protein is now phosphorylated. Step 3. The phosphorylated protein undergoes a conformational change, exposing the three Na+ to the outside. In this conformation, the pump has a low affinity for Na+, allowing the three bound Na+ ions to diffuse into the extracellular fluid. Step 4. The phosphorylated protein has a high affinity for K+, two of which bind to the extracellular side of the pump after Na+ diffuses away. Step 5. The binding of the K+ causes hydrolysis of the bound phosphate, and a conformational change back to the binding pocket facing the cytoplasm. Step 6. The pump binds ATP, returning to a state with a low affinity for K+ and high affinity for Na+. The bound K+ diffuse away, and three new Na+ can bind to initiate another cycle. In every cycle, three Na+ leave the cell and two K+ enter. The changes in protein conformation that occur during the cycle are rapid, enabling each carrier to transport as many as 300 Na+ per second. The Na+/K+ pump appears to exist in all animal cells, although cells vary widely in the number of pump proteins they contain.

Coupled transport uses ATP indirectly Some molecules are moved against their concentration gradient by using the energy stored in a gradient of a different molecule. In this process, called coupled transport, the energy released as one molecule moves down its concentration gradient is captured and used to move a different molecule against its gradient. As you just saw, the energy stored in ATP molecules can be used to create a gradient of Na+ and K+ across the membrane. These gradients can then be used to power the transport of other molecules across the membrane. As one example, let’s consider the active transport of glucose across the membrane in animal cells. Glucose is such an important molecule that there are a variety of transporters for it, one of which was discussed in section 5.4 under passive transport. In a multicellular organism, intestinal epithelial cells can have a higher concentration of glucose inside the cell than outside, so

Extracellular

Na+ K+

ATP

P

Intracellular ATP

1. ATP and 3 Na+ ions bind to pump.

6. Protein returns to original conformation with low affinity for K+ and K+ diffuses away. ATP can bind to start the cycle again.

+

ADP

ATP 2. Bound ATP is used to phosphorylate pump.

P P

P P 5. Binding of potassium causes dephosphorylation of protein.

4. This conformation has higher affinity for K+. Extracellular potassium binds to exposed sites.

3. Phosphorylation causes conformational change in protein, reducing its affinity for Na+. The Na+ then diffuses out.

Figure 5.14 The sodium–potassium pump. The sodium–potassium pump is a protein carrier that transports sodium (Na+) and potassium

(K+) across the plasma membrane. For every three Na+ transported out of the cell, two K+ are transported into it. Energy for the process is provided by ATP hydrolysis. The pump changes conformation between high affinity for Na+ and high affinity for K+ based on phosphorylation state.

these cells need to be able to transport glucose against its concentration gradient. This requires energy and a different transporter from the one involved in facilitated diffusion of glucose. The active glucose transporter uses the Na+ gradient produced by the Na+/K+ pump as a source of energy to power the movement of glucose into the cell. In this system, both glucose and Na+ bind to the transport protein, which allows Na+ to pass into the cell down its concentration gradient, capturing the energy and using it to move glucose into the cell. In this kind of cotransport, both molecules are moving in the same direction across the membrane; therefore the transporter is a symporter (figure 5.15). In a related process, called countertransport, the inward movement of Na+ is coupled with the outward movement of another substance, such as Ca2+ or H+. As in cotransport, both Na+ and the other substance bind to the same transport protein, which in this case is an antiporter, as the substances bind on opposite sides of the membrane and are moved in opposite directions. In countertransport, the cell uses the energy released as Na+ moves down its concentration gradient into the cell to

eject a substance against its concentration gradient. In both cotransport and countertransport, the potential energy in the concentration gradient of one molecule is used to transport another molecule against its concentration gradient. They differ only in the direction in which the second molecule moves relative to the first.

Learning Outcomes Review 5.5

Active transport requires both a carrier protein and energy, usually in the form of ATP, to move molecules against a concentration gradient. The Na+/K+ pump uses ATP to move Na+ in one direction and K+ in the other to create and maintain concentration differences of these ions. In coupled transport, a favorable concentration gradient of one molecule is used to move a different molecule against its gradient, such as in the transport of glucose by Na+. ■■

Can active transport involve a channel protein? Why or why not?

chapter

5 Membranes 105

Outside of cell

Na+

Glucose Coupled transport protein

Na+/ K+ pump

matter (figure 5.16a), the process is called phagocytosis (Greek phagein, “to eat,” + cytos, “cell”). If the material the cell takes in is liquid (figure 5.16b), the process is called pinocytosis (Greek pinein, “to drink”). Pinocytosis is common among animal cells. Mammalian egg cells, for example, “nurse” from surrounding cells; the nearby cells secrete nutrients that the maturing egg cell takes up by pinocytosis. Virtually all eukaryotic cells constantly carry out these kinds of endocytotic processes, trapping particles and extracellular fluid in vesicles and ingesting them. Endocytosis rates vary from one cell type to another. They can be surprisingly high; some types of white blood cells ingest up to 25% of their cell volume each hour.

ATP

Receptor-mediated endocytosis

ADP + Pi

Inside of cell

K+

Figure 5.15 Coupled transport. A membrane protein

transports Na+ into the cell, down its concentration gradient, at the same time it transports a glucose molecule into the cell. The gradient driving the Na+ entry allows sugar molecules to be transported against their concentration gradient. The Na+ gradient is maintained by the Na+/K+ pump. ADP = adenosine diphosphate; ATP = adenosine triphosphate; P i = inorganic phosphate

5.6

Bulk Transport by Endocytosis and Exocytosis

Learning Outcomes 1. Distinguish between endocytosis and exocytosis. 2. Illustrate how endocytosis can be specific.

The lipid nature of cell plasma membranes raises a second problem. The substances cells require for growth are mostly large, polar molecules that cannot cross the hydrophobic barrier a lipid bilayer creates. How do these substances get into cells? Two processes are involved in this bulk transport: endocytosis and exocytosis.

Bulk material enters the cell in vesicles In endocytosis, the plasma membrane envelops food particles and fluids. Cells use three major types of endocytosis: phago­cytosis, pinocytosis, and receptor-mediated endocytosis (figure 5.16). Like active transport, these processes also require energy expenditure.

Phagocytosis and pinocytosis If the material the cell takes in is particulate (made up of discrete particles), such as an organism or some other fragment of organic 106 part II Biology of the Cell

Molecules are often transported into eukaryotic cells through receptor-mediated endocytosis. These molecules first bind to specific receptors in the plasma membrane—they have a conformation that fits snugly into the receptor. Different cell types contain a characteristic battery of receptor types, each for a different kind of molecule in their membranes. The portion of the receptor molecule that lies inside the membrane is trapped in an indented pit coated on the cytoplasmic side with the protein clathrin. Each pit acts like a molecular mousetrap, closing over to form an internal vesicle when the right molecule enters the pit (figure 5.16c). The trigger that releases the trap is the binding of the properly fitted target molecule to the embedded receptor. When binding occurs, the cell reacts by initiating endocytosis; the process is highly specific and very fast. The vesicle is now inside the cell carrying its cargo. One type of molecule that is taken up by receptor-mediated endocytosis is low-density lipoprotein (LDL). LDL molecules bring cholesterol into the cell where it can be incorporated into membranes. Cholesterol plays a key role in determining the stiffness of the body’s membranes. In the human genetic disease familial hypercholesterolemia, the LDL receptors lack tails, so they are never fastened in the clathrin-coated pits and, as a result, do not trigger vesicle formation. The cholesterol stays in the bloodstream of affected individuals, accumulating as plaques inside arteries and leading to heart attacks. It is important to understand that endocytosis in itself does not bring substances directly into the cytoplasm of a cell. The material taken in is still separated from the cytoplasm by the membrane of the vesicle.

Material can leave the cell by exocytosis The reverse of endocytosis is exocytosis, the discharge of material from vesicles at the cell surface (figure 5.17). In plant cells, exocytosis is an important means of exporting the materials needed to construct the cell wall through the plasma membrane. Among protists, contractile vacuole discharge is considered a form of exocytosis. In animal cells, exocytosis provides a mechanism for secreting many hormones, neurotransmitters, digestive enzymes, and other substances. The mechanisms for transport across cell membranes are summarized in table 5.2.

Figure 5.16 Endocytosis. Both Bacterial (a) phagocytosis and (b) pinocytosis are forms of cells endocytosis. c. In receptor-mediated endocytosis, cells have pits coated with the protein clathrin Plasma that initiate endocytosis when target molecules membrane bind to receptor proteins in the plasma membrane. Photo inserts (false color has been added to enhance distinction of structures): (a) A a. Phagocytosis TEM of phagocytosis of a bacterium, Rickettsia tsutsugamushi, by a mouse peritoneal mesothelial cell. The bacterium enters the host cell by Solute phagocytosis and replicates in the cytoplasm. (b) A TEM of pinocytosis in a smooth muscle Plasma cell. (c) A coated pit appears in the plasma membrane membrane of a developing egg cell, covered with a layer of proteins. When an appropriate collection of molecules gathers in the coated pit, b. Pinocytosis the pit deepens and will eventually seal off to form a vesicle. (a): Dr. Edwin P. Ewing, Jr./CDC;

(b): Don W. Fawcett/Science Source; (c) top/bottom: Don W. Fawcett/Science Source

Cytoplasm 1 μm

Cytoplasm 0.1 μm

Target molecule

Receptor protein Coated pit

Learning Outcomes Review 5.6

Large molecules and other bulky materials can enter a cell by endocytosis and leave the cell by exocytosis. These processes require energy. Endocytosis may be mediated by specific receptor proteins in the membrane that trigger the formation of vesicles. ■■

Coated vesicle

Clathrin

c. Receptor-mediated endocytosis

90 nm

What feature unites transport by receptor-mediated endocytosis, transport by a carrier, and catalysis by an enzyme?

Plasma membrane Secretory product

Secretory vesicle Cytoplasm

a.

b.

70 nm

Figure 5.17 Exocytosis. a. Proteins and other molecules are secreted from cells in small packets called vesicles, whose membranes fuse with the plasma membrane, releasing their contents outside the cell. b. A false-colored transmission electron micrograph showing exocytosis. (c): Dr. Birgit Satir

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5 Membranes 107

TA B L E 5 . 2 Process

Mechanisms for Transport Across Cell Membranes How It Works

Example

PASSIVE PROCESSES Diffusion Direct

Random molecular motion produces net migration of nonpolar molecules toward region of lower concentration

Movement of oxygen into cells

Protein channel

Polar molecules or ions move through a protein channel; net movement is toward region of lower concentration

Movement of ions in or out of cell

Protein carrier

Molecule binds to carrier protein in membrane and is transported across; net movement is toward region of lower concentration

Movement of glucose into cells

Diffusion of water across the membrane via osmosis; requires osmotic gradient

Movement of water into cells placed in a hypotonic solution

Facilitated Diffusion

Osmosis Aquaporins

ACTIVE PROCESSES Active Transport Protein carrier Na+/K+ pump

Carrier uses energy to move a substance across a membrane against its concentration gradient

Na+ and K+ against their concentration gradients

Coupled transport

Molecules are transported across a membrane against their concentration gradients by the cotransport of sodium ions or protons down their concentration gradients

Coupled uptake of glucose into cells against its concentration gradient using a Na+ gradient

Phagocytosis

Particle is engulfed by membrane, which folds around it and forms a vesicle

Ingestion of bacteria by white blood cells

Pinocytosis

Fluid droplets are engulfed by membrane, which forms vesicles around them

“Nursing” of human egg cells

Receptor-mediated endocytosis

Endocytosis triggered by a specific receptor, forming clathrin-coated vesicles

Cholesterol uptake

Vesicles fuse with plasma membrane and eject contents

Secretion of mucus; release of neurotransmitters

Endocytosis Membrane vesicle

Exocytosis Membrane vesicle

108 part II Biology of the Cell

Chapter Review Keith R. Porter/Science Source

5.1 The Structure of Membranes The fluid mosaic model shows proteins embedded in a fluid lipid bilayer. Membranes are sheets of phospholipid bilayers with associated proteins (figure 5.3). Hydrophobic regions of a membrane are oriented inward and hydrophilic regions oriented outward. In the fluid mosaic model, proteins float on or in the lipid bilayer. Cellular membranes consist of four component groups. In eukaryotic cells, membranes have four components: a phospholipid bilayer, transmembrane proteins (integral membrane proteins), an interior protein network, and cell-surface markers. The interior protein network is composed of cytoskeletal filaments and peripheral membrane proteins, which are associated with the membrane but are not an integral part of it. Membranes contain glycoproteins and glycolipids on the surface that act as cell identity markers. Cholesterol and sphingolipid can associate to form microdomains. The two leaflets of the plasma membrane are also not identical. Electron microscopy has provided structural evidence. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have confirmed the structure predicted by the fluid mosaic model.

5.2 Phospholipids: The Membrane’s Foundation Phospholipids are composed of two fatty acids and a phosphate group linked to a three-carbon glycerol molecule. Phospholipids spontaneously form bilayers. The phosphate group of a phospholipid is polar and hydrophilic; the fatty acids are nonpolar and hydrophobic, and they orient away from the polar head of the phospholipids. The nonpolar interior of the lipid bilayer impedes the passage of water and water-soluble substances. The phospholipid bilayer is fluid. Hydrogen bonding of water keeps the membrane in its bilayer configuration; however, phospholipids and unanchored proteins in the membrane are loosely associated and can diffuse laterally. Membrane fluidity varies with lipid composition. Membrane fluidity depends on the fatty acid composition of the membrane. Unsaturated fats tend to make the membrane more fluid because of the “kinks” of double bonds in the fatty acid tails. Temperature also affects fluidity. Phospholipid composition affects membrane structure. Different membrane compartments have different phospholipid composition. This affects both structure and function of different membranes, and persists despite membrane traffic.

5.3 Proteins: Multifunctional Components

may cross the bilayer a number of times, and each membrane-spanning region is called a transmembrane domain. Such a domain is composed of hydrophobic amino acids usually arranged in α helices. In certain proteins, β-pleated sheets in the nonpolar region form a pipelike passageway having a polar environment. An example is the porin class of proteins.

5.4 Passive Transport Across Membranes Transport can occur by simple diffusion. Simple diffusion is the passive movement of a substance along a chemical or electrical gradient. Biological membranes pose a barrier to hydrophilic polar molecules, while they allow hydrophobic substances to diffuse freely. Proteins allow membrane diffusion to be selective. Ions and large hydrophilic molecules cannot cross the phospholipid bilayer. Diffusion can still occur via channel or carrier proteins by facilitated diffusion. Channels allow the diffusion of specific ions, by forming an aqueous pore in the membrane. Carrier proteins bind to the molecules they transport, much as enzymes do. The rate of transport by a carrier is limited by the number of carriers in the membrane. Osmosis is the movement of water across membranes. The direction of movement due to osmosis depends on the solute concentration on either side of the membrane (figures 5.12 and 5.13). Solutions can be isotonic, hypotonic, or hypertonic. Cells in an isotonic solution are in osmotic balance; cells in a hypotonic solution will gain water; and cells in a hypertonic solution will lose water. Aquaporins are water channels that facilitate the diffusion of water.

5.5 Active Transport Across Membranes Active transport uses energy to move materials against a concentration gradient. Active transport uses specialized protein carriers that couple a source of energy to transport. They are classified based on the number of molecules and direction of transport. Uniporters transport a specific molecule in one direction; symporters transport two molecules in the same direction; and antiporters transport two molecules in opposite directions. The sodium–potassium pump runs directly on ATP. The sodium–potassium pump moves Na+ out of the cell and K+ into the cell against their concentration gradients using ATP. In every cycle of the pump, three Na+ leave the cell and two K+ enter it. This pump appears to be almost universal in animal cells. Coupled transport uses ATP indirectly. Coupled transport uses a concentration gradient of one molecule to move another against a gradient in the same direction. Countertransport is similar, but the two molecules move in opposite directions.

Proteins and protein complexes perform key functions. Transporters are integral membrane proteins that carry specific substances through the membrane. Enzymes often occur on the interior surface of the membrane. Cell-surface receptors respond to external chemical messages and change conditions inside the cell; cell identity markers on the surface allow recognition of the body’s cells as “self.” Cell-to-cell adhesion proteins glue cells together; surface proteins that interact with other cells anchor to the cytoskeleton.

5.6 Bulk Transport by Endocytosis and Exocytosis

Structural features of membrane proteins relate to function. Surface proteins are attached to the surface by nonpolar regions that associate with polar regions of phospholipids. Transmembrane proteins

Material can leave the cell by exocytosis. In exocytosis, material in a vesicle is discharged when the vesicle fuses with the membrane.

Bulk transport moves large quantities of substances that cannot pass through the cell membrane. Bulk material enters the cell in vesicles. In endocytosis, the cell membrane surrounds material and pinches off to form a vesicle. In receptor-mediated endocytosis, specific molecules bind to receptors on the cell membrane.

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5 Membranes 109

Visual Summary Fluid mosaic model

Consists of

Cellular membranes

mediate

Transport 3 types

Glycerol, 2 fatty acids, phosphate made of

Forms spontaneously

Phospholipid bilayer Transmembrane proteins

Fluid include

Passive transport 3 types

Transporters Enzymes

Cytoskeleton

Receptors Cell surface markers Function Cell recognition

types

move down

Concentration gradient

Concentration difference

Osmosis Movement of water

Facilitated diffusion is

Adhesion proteins

Selective

Cytoskeleton attachment

Active transport

Bulk transport types

defined as

Simple diffusion

Identity markers

move up

example Sodiumrequires potassium pump

Carrier proteins uses Channel proteins

Movement into the cell

Endocytosis is specific

Energy Carrier proteins

Glycoproteins

Receptormediated endocytosis

Exocytosis

Movement out of the cell

Glycolipids

Review Questions Keith R. Porter/Science Source

U N D E R S TA N D 1. The fluid mosaic model of the membrane describes the membrane as a. b. c. d.

containing a significant quantity of water in the interior. composed of fluid phospholipids on the outside and protein on the inside. composed of protein on the outside and fluid phospholipids on the inside. made of proteins and lipids that can freely move.

2. What chemical property characterizes the interior of the phospholipid bilayer? a. b. c. d.

It is hydrophobic. It is hydrophilic. It is polar. It is saturated.

3. The transmembrane domain of an integral membrane protein a. b.

is composed of hydrophobic amino acids. often forms an α-helical structure.

110 part II Biology of the Cell

c. d.

can cross the membrane multiple times. All of the choices are correct.

4. The specific function of a membrane within a cell is determined by the a. b. c. d.

degree of saturation of the fatty acids within the phospholipid bilayer. location of the membrane within the cell. presence of lipid rafts and cholesterol. type and number of membrane proteins.

5. The movement of water across a membrane is dependent on a. b. c. d.

the solvent concentration. the solute concentration. the presence of carrier proteins. membrane potential.

6. Cells immersed in a solution of salt and sugar respond by shrinking. From this we would infer that: a. b.

the solution is isotonic. the solution is hypertonic.

c. the solution is hypotonic. d. we know nothing about the solution. 7. Which of the following is NOT a mechanism for bringing material into a cell? a. Exocytosis b. Endocytosis c. Pinocytosis d. Phagocytosis

A P P LY 1. A bacterial cell that can alter the composition of saturated and unsaturated fatty acids in its membrane lipids is adapted to a cold environment. If this cell is shifted to a warmer environment, it will react by a. b. c. d.

increasing the amount of cholesterol in its membrane. altering the amount of protein present in the membrane. increasing the degree of saturated fatty acids in its membrane. increasing the percentage of unsaturated fatty acids in its membrane.

2. What variable(s) influence(s) whether a nonpolar molecule can move across a membrane by passive diffusion? a. The structure of the phospholipids bilayer b. The difference in concentration of the molecule across the membrane c. The presence of transport proteins in the membrane d. All of the choices are correct. 3. Which of the following does NOT contribute to the selective permeability of a biological membrane? a. b. c. d.

Specificity of the carrier proteins in the membrane Selectivity of channel proteins in the membrane Hydrophobic barrier of the phospholipid bilayer Hydrogen bond formation between water and phosphate groups

4. How are active transport and coupled transport related? a. They both use ATP to move molecules. b. Active transport establishes a concentration gradient, but coupled transport doesn’t.

c.

Coupled transport uses the concentration gradient established by active transport. d. Active transport moves one molecule, but coupled transport moves two. 5. A cell can use the process of facilitated diffusion to a. concentrate a molecule such as glucose inside a cell. b. remove all of a toxic molecule from a cell. c. move ions or large polar molecules across the membrane regardless of concentration. d. move ions or large polar molecules from a region of high concentration to a region of low concentration.

SYNTHESIZE 1. Figure 5.6 describes a classic experiment demonstrating the ability of proteins to move within the plane of the cell’s plasma membrane. The following table outlines three different experiments using the fusion of labeled mouse and human cells. Experiment

Conditions

Temperature (°C)

Result

1

Fuse human and mouse cells

37

Intermixed membrane proteins

2

Fuse human and mouse cells in presence of ATP inhibitors

37

Intermixed membrane proteins

3

Fuse human and mouse cells

4

No intermixing of membrane proteins

What conclusions can you reach about the movement of these proteins? 2. Each compartment of the endomembrane system of a cell is connected to the plasma membrane. Create a simple diagram of a cell, including the RER, Golgi apparatus, vesicle, and plasma membrane. Starting with the RER, use two different colors to represent the inner and outer halves of the bilayer for each of these membranes. What do you observe? 3. The distribution of lipids in the ER membrane is symmetric, that is, it is the same in both leaflets of the membrane. The Golgi apparatus and plasma membrane do not have symmetric distribution of membrane lipids. What kinds of processes could achieve this outcome?

chapter

5 Membranes 111

6

CHAPTER

Energy and Metabolism Chapter Contents 6.1

The Flow of Energy in Living Systems

6.2

The Laws of Thermodynamics and Free Energy

6.3

ATP: The Energy Currency of Cells

6.4

Enzymes: Biological Catalysts

6.5

Metabolism: The Chemical Description of Cell Function

Robert Caputo/Aurora Photos/Cavan images

Introduction

Visual Outline Metabolism

Energy can be either

is the Ability to do work

Kinetic

or

Potential

Thermodynamics

1st law

requires

involves

Cellular chemical reactions

Enzymes

2nd law

types

are Biological catalysts

Specific

Anabolic

Catabolic

Build up

Break down

Life can be viewed as a constant flow of energy, channeled by organisms to do work through living. Each of the significant properties by which we define life—order, growth, reproduction, responsiveness, and internal regulation—requires a constant supply of energy. Energy that the lion extracts from its meal of a giraffe will be used to run its cells, power its roar, fuel its running, and build a bigger lion. Deprived of a source of energy, life stops. Therefore, a comprehensive study of life would be impossible without discussing bioenergetics, the analysis of how energy powers the activities of living systems. In this chapter, we focus on energy—what it is and how it changes during chemical reactions.

Energy can take many forms: mechanical energy, heat, sound, electric current, light, or radioactivity. Because it can exist in so many forms, energy can be measured in many ways. Heat is the most convenient way of measuring energy because all other forms of energy can be converted into heat. In fact, the term thermodynamics means “heat changes.” The unit of heat most commonly employed in biology is the kilocalorie (kcal). One kilocalorie is equal to 1000 calories (cal). One calorie is the heat required to raise the temperature of one gram of water one degree Celsius (°C). (You are probably more used to seeing the term Calorie with a capital C. This is used on food labels and is actually the same as kilocalorie.) Another energy unit, often used in physics, is the joule; one joule equals 0.239 cal.

6.1 The

Flow of Energy in Living Systems

Learning Outcomes 1. Differentiate between kinetic and potential energy. 2. Identify the source of energy for the biosphere. 3. Describe the nature of redox reactions.

Thermodynamics is the branch of chemistry concerned with ­energy changes. Cells are governed by the laws of physics and chemistry, so we must understand these laws in order to understand how cells function.

Energy can take many forms Energy is defined as the capacity to do work. We think of energy­ as existing in two states: kinetic energy and potential energy (figure 6.1). Kinetic energy is the energy of motion. Moving objects perform work by causing other matter to move. Potential ­energy is stored energy. Objects that are not actively moving but have the capacity to do so possess potential energy. A boulder perched on a hilltop has gravitational potential energy. As it begins to roll downhill, some of its potential energy is converted into kinetic energy. Much of the work that living organisms carry out involves transforming potential energy into kinetic energy.

a. Potential energy

The Sun provides energy for most living systems Energy flows into most ecosystems from the Sun. It is estimated that sunlight provides the Earth with more than 1.3 × 1024 calories per year, or 4 × 1016 calories per second! Plants, algae, and certain kinds of bacteria capture a fraction of this energy through photosynthesis. There are also ecosystems where bacteria extract energy from the oxidation of inorganic compounds. As we will see, these initial sources of energy literally power the energetic needs of all living systems. In chemical reactions, breaking chemical bonds requires energy, and forming covalent bonds releases energy. If a reaction forms a more ordered, higher-energy state, as photosynthesis does, an input of energy is required. In photosynthesis, energy absorbed from sunlight is used to combine small molecules (water and carbon dioxide) into more complex ones (sugars). In the

b. Kinetic energy

Figure 6.1 Potential and kinetic energy. a. Objects that have the capacity to move but are not moving have potential energy. The energy required for the girl to climb to the top of the slide is stored as potential energy. b. Objects that are in motion have kinetic energy. The stored potential energy is released as kinetic energy as the girl slides down. chapter

6 Energy and Metabolism 113

Loss of electron (oxidation)

6.2 The

e− A

+

A

B

B

A+

+

B−

Gain of electron (reduction) lower energy

higher energy

Figure 6.2 Redox reactions. Oxidation is the loss of an

electron; reduction is the gain of an electron. In this example, the charges of molecules A and B appear as superscripts in each molecule. Molecule A loses energy as it loses an electron, and molecule B gains that energy as it gains an electron.

process, energy from sunlight is stored as potential energy in the structure of the sugar molecules.

Redox reactions transfer electrons During a chemical reaction, the energy stored in chemical bonds may be used to make new bonds. One important class of reactions involves the transfer of one or more electrons from one atom or molecule to another. The atom or molecule that loses an electron is said to be oxidized, and the process is called oxidation. The atom or molecule that gains an electron is said to be reduced, and the process is called reduction. The reduced form of a molecule has a higher level of potential energy than the oxidized form (figure 6.2). Oxidation and reduction always take place together, because every electron that is lost by one atom through oxidation is gained by another atom through reduction. Therefore, chemical reactions of this sort are called ­oxidation–reduction, or redox, reactions. Redox reactions transfer energetic electrons when bonds are made or broken, and with these electrons the potential energy that the electrons hold. It is for this reason that the reduced form of the molecule has a higher level of energy than the oxidized form. Oxidation–reduction reactions play a key role in the flow of energy through biological systems. In the next two chapters, you will learn the details of how organisms derive energy from the oxidation of organic compounds via respiration, as well as from the energy in sunlight via photosynthesis.

Learning Outcomes Review 6.1

Energy is defined as the capacity to do work. The two forms of energy are kinetic energy, or energy of motion, and potential energy, or stored energy. The ultimate source of energy for living systems is the Sun. Organisms derive their energy from redox reactions. In oxidation, a molecule loses an electron; in reduction, a molecule gains an electron. ■■

What energy source might ecosystems at the bottom of the ocean use?

114 part II Biology of the Cell

Laws of Thermodynamics and Free Energy

Learning Outcomes 1. Explain the laws of thermodynamics. 2. Relate free energy changes to the outcome of chemical reactions. 3. Contrast the course of a reaction with and without an enzyme catalyst.

Thermodynamics is the branch of chemistry concerned with ­energy changes. Cells are governed by the laws of physics and chemistry, so we must understand these laws in order to understand how cells function. All activities of living organisms—growing, ­running, thinking, singing, reading these words—involve changes in energy. Two universal laws, which we call the laws of thermodynamics, govern all energy changes in the universe, from the nuclear reactions that occur in the sun, to your picking up this book.

The First Law states that energy cannot be created or destroyed The First Law of Thermodynamics concerns the amount of energy in the universe. Energy cannot be created or destroyed; it can only change from one form to another (from potential to kinetic, for example). The total amount of energy in the universe remains constant. The lion eating a giraffe at the beginning of this chapter is acquiring energy. Rather than creating new energy, the lion is transferring some of the potential energy stored in the giraffe’s tissues to its own body, just as the giraffe obtained the potential energy stored in the plants it ate while it was alive, and those plants captured energy from sunlight. Within any living organism, chemical potential energy stored in some molecules can be shifted to other molecules and stored in different chemical bonds. It can also be converted into other forms, such as kinetic energy, light, or electricity. During each conversion, some of the energy dissipates into the environment as heat, which is a measure of the random motion of molecules (and therefore a measure of one form of kinetic energy). Energy continuously flows through the biological world in one direction, with new energy from the Sun constantly entering the system to replace the energy dissipated as heat. Heat can be harnessed to do work only when there is a heat gradient—that is, a temperature difference between two areas. Cells are too small to maintain significant internal temperature differences, so heat energy is incapable of doing the work of cells. Instead, cells must rely on chemical reactions for energy. Although the total amount of energy in the universe remains constant, the energy available to do work decreases as more of it is progressively lost as heat.

The Second Law states that some energy is lost as disorder increases The Second Law of Thermodynamics addresses the efficiency of energy transformations. Energy cannot be transformed from one form to another with 100% efficiency; some energy is always unavailable. This unavailable energy manifests as an increase in the random motion of molecules, an increase in the number of energy states available to atoms, or increased dispersal of energy in the system. This is often characterized as an increase in the randomness or disorder of the system. We measure this as an increase in entropy (symbolized by S in the equations in the next section). Put simply, disorder is more likely than order. For example, it is more likely that a column of bricks will fall over than that a pile of bricks will spontaneously form a column. Energy transformations proceed spontaneously to convert matter from a more ordered, less stable form to a less ordered, but more stable form. Consider a glass of ice water in a room at 25°C. The water molecules in the ice form an ordered, regular array, while the water molecules in the liquid are more disordered. There are far fewer ways that the molecules can be arranged in the crystal than in the liquid water. As the ice absorbs heat from the surroundings, the hydrogen bonds holding the crystal together are broken and the water molecules become less ordered (higher entropy). When all of the ice has melted, the entropy of the water will be maximal for this system (figure 6.3). You can order the images in figure 6.3 based on experience, but the underlying reason is the Second Law. For this reason, it is sometimes called time’s arrow.

Chemical reactions can be predicted based on changes in free energy It takes energy to break the chemical bonds that hold the atoms in a molecule together. Heat energy, because it increases atomic motion, makes it easier for the atoms to pull apart. For this reason, both chemical bonding and heat have a significant thermodynamic influence on a molecule, the former reducing disorder and the latter increasing it. The net effect, the amount of energy actually available to break and subsequently form other chemical bonds, is called the free energy of that molecule.

Figure 6.3 Entropy increases as ice melts. The entropy of

the water molecules increases as the ice melts. The number of molecular states that each molecule can occupy increases as the hydrogen bonds holding the molecules in a crystalline lattice in the ice are broken. The molecules in the liquid water are continually breaking and re-forming hydrogen bonds.

We define free energy as the energy available to do work at constant temperature in a system. For conditions of constant temperature, pressure, and volume, we call this Gibbs free energy, symbolized by G. G is equal to the total energy in a molecule’s chemical bonds (called enthalpy and designated H) minus a term (TS) that expresses the amount of disorder in the system, where S is the entropy and T is the absolute temperature in degrees Kelvin (K = °C + 273): G = H – TS Chemical reactions may break some bonds in the reactants and form new bonds in the products. Consequently, reactions can produce changes in free energy. When a chemical reaction occurs under conditions of constant temperature, pressure, and volume—as do most biological reactions—the change (symbolized by the Greek capital letter delta, Δ) in free energy is simply ΔG = ΔH – TΔS We can use the change in free energy to predict whether a particular chemical reaction is spontaneous or not. The change in free energy is calculated as energy of products minus energy of reactants, so if the products have more free energy than the reactants, ΔG is positive. Such reactions are not spontaneous because they require an input of energy. This can be because either the bond energy (H) is higher, or the disorder (S) in the system is ­lower. We call this an endergonic reaction. A plot of free energy for reactants and products shows this graphically (figure 6.4a). An endergonic reaction is “uphill” from reactants to products, and not spontaneous. Conversely, if the products have less free energy than the reactants, ΔG is negative, and the reaction will proceed spontaneously. The negative ΔG can be because the bond energy (H) is lower, or the disorder (S) is higher, or both. These reactions release energy and are called exergonic reactions. The plot of free energy for the reaction is now “downhill” from reactants to products, and the reaction will be spontaneous (figure 6.4b). Note that spontaneous does not mean the same thing as instantaneous. A spontaneous reaction may proceed very slowly. Because chemical reactions are reversible, a reaction that is exergonic in the forward direction will be endergonic in the reverse direction. For each reaction, an equilibrium exists between the relative amounts of reactants and products. This can be expressed quantitatively by the equilibrium constant (Keq), a ratio of the concentrations of products divided by concentrations of reactants. For exergonic reactions, ΔG will be negative, and Keq will be >1, meaning equilibrium favors the products. For endergonic reactions ΔG will be positive, and Keq will be 0 Course of Reaction

Progress of Reaction

a.

Free Energy (G)

ΔG

Figure 6.5 Activation energy and catalysis. Exergonic reactions do not necessarily proceed rapidly because activation energy must be supplied to destabilize existing chemical bonds. Catalysts accelerate particular reactions by lowering the amount of activation energy required to initiate the reaction. Catalysts do not alter the free-energy change produced by the reaction.

Reactants

Products

Energy is released ΔG < 0

Progress of Reaction

b.

Figure 6.4 Energy in chemical reactions.  a. In an endergonic reaction, the products of the reaction contain more energy than the reactants, and the extra energy must be supplied for the reaction to proceed. b. In an exergonic reaction, the products contain less energy than the reactants, and the excess energy is released.

not spontaneously explode. One reason is that most reactions require an input of energy to get started. In the case of your car, this input consists of the electrical sparks in the engine’s cylinders, producing a controlled explosion.

Activation energy Before new chemical bonds can form, even bonds that contain less energy, existing bonds must first be broken, and that requires energy input. The extra energy needed to destabilize existing chemical bonds and initiate a chemical reaction is called activation energy (figure 6.5). The rate of an exergonic reaction depends on the activation energy required for the reaction to begin. Reactions with larger activation energies tend to proceed more slowly because fewer molecules will collide with sufficient energy to get over the initial energy hurdle. The rate of reactions can be increased in two ways: (1) by increasing the energy of reacting molecules or (2) by lowering activation energy. Chemists often drive important industrial reactions by increasing the energy of the reacting molecules, which is frequently accomplished simply by heating up the 116 part II Biology of the Cell

reactants. The other strategy is to use a catalyst to lower the activation energy.

How catalysts work Stressing particular chemical bonds can make them easier to break. The process of influencing chemical bonds in a way that lowers the activation energy needed to initiate a reaction is called catalysis, and substances that accomplish this are known as catalysts (figure 6.5). Catalysts exert their action by affecting an intermediate stage in a reaction—the transition state. The energy needed to reach this transition state is the activation energy. Catalysts stabilize this transition state, thus lowering activation energy. Catalysts cannot violate the basic laws of thermo­dynamics; they cannot make an endergonic reaction proceed spontaneously. By reducing the activation energy, a catalyst accelerates both the forward and the reverse reactions by exactly the same amount. Therefore, a catalyst does not alter the proportion of reactant ultimately converted into product. To understand this, imagine a bowling ball resting in a shallow depression on the side of a hill. Only a narrow rim of dirt below the ball prevents it from rolling down the hill. Now imagine digging away that rim of dirt. If you remove enough dirt from below the ball, it will start to roll down the hill—but removing dirt from below the ball will never cause the ball to roll up the hill. Removing the lip of dirt simply allows the ball to move freely; gravity determines the direction it then travels. Similarly, the direction in which a chemical reaction proceeds is determined solely by the difference in free energy between reactants and products. Like digging away the soil below the bowling ball on the hill, catalysts reduce the energy barrier that is preventing the reaction from proceeding. Only exergonic­reactions can proceed spontaneously, and catalysts cannot change that. What catalysts can do is make a reaction proceed much faster. In living systems, enzymes act as catalysts.

The First Law of Thermodynamics states that energy cannot be created or destroyed. The Second Law states that disorder, or entropy, is increasing. Free-energy changes (∆G) can predict whether chemical reactions take place. Reactions with a negative ∆G occur spontaneously, and those with a positive ∆G do not. Energy needed to initiate a reaction is termed activation energy. Catalysts stabilize an intermediate transition state, lowering activation energy and accelerating reactions.

Triphosphate group O−

Can an enzyme make an endergonic reaction exergonic?

O

6.3 ATP:

The Energy Currency of Cells

P

O−

O

ADP

■■

ATP

Learning Outcomes Review 6.2

O

P

a.

High-energy bonds O− Adenine N

O AMP CORE

Learning Outcomes 1. Describe the role of ATP in short-term energy storage. 2. Distinguish which bonds in ATP are “high energy.”

In chapter 3 we introduced adenosine triphosphate (ATP) as both a precursor for RNA and also the energy currency for the cell. We call ATP the energy currency, because the energy released by ATP hydrolysis can power most endergonic transactions.

Cells store and release energy in the bonds of ATP The process of energy metabolism in cells is complex, as we shall see in the next two chapters. To provide energy when needed, cells use energy released by the exergonic reactions of energy metabolism to synthesize an intermediate that can later be degraded to release energy on demand. The molecule that has evolved to fulfill this role in cells is ATP.

The structure of ATP ATP is composed of three smaller components (figure 6.6). The first component is a 5-carbon sugar, ribose, which serves as the framework to which the other two subunits are attached. The second component is adenine, an organic molecule composed of two carbon–nitrogen rings. Each of the nitrogen atoms in the ring has an unshared pair of electrons and weakly attracts hydrogen ions, making adenine chemically a weak base. The third component of ATP is a chain of three phosphates, thus adenosine triphosphate.

How ATP stores energy The key to how ATP stores energy lies in its triphosphate group. Phosphate groups are highly negatively charged, and thus they strongly repel one another. This electrostatic repulsion makes the covalent bonds joining the phosphates unstable. The molecule is

H C N

O

P O

NH2 C

C

C

N C

H

N

O− O

CH2 H

H

H

OH

OH

H

Ribose

b.

Figure 6.6 The ATP molecule. The model (a) and the structural diagram (b) both show that ATP has a core of AMP. Addition of one phosphate to AMP yields ADP, and addition of a second phosphate yields ATP. These two terminal phosphates are attached by high-energy bonds so that removing either by hydrolysis is an exergonic reaction that releases energy. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate. often referred to as a “coiled spring,” with the phosphates straining away from one another. The unstable bonds holding the phosphates together in the ATP molecule have a low activation energy and are easily broken by hydrolysis. This reaction is exergonic, releasing a c­onsiderable amount of energy. In other words, the hydrolysis of ATP has a negative ΔG, and the energy it releases can be used to perform work. For this reason, we often refer to these as “high-energy” bonds, referring to this release of energy, not to the actual bond energy. In most reactions involving ATP, only the outermost highenergy phosphate bond is hydrolyzed, cleaving off the phosphate group on the end. When this happens, ATP becomes aden­o­sine diphosphate (ADP) plus an inorganic phosphate (Pi), and e­ nergy chapter

6 Energy and Metabolism 117

equal to 7.3 kcal/mol is released under standard conditions. This reaction can also transfer the terminal phosphate to another molecule. When this is hydrolyzed, it will release Pi. Both of the two terminal phosphates can be hydrolyzed to release energy, leaving adenosine monophosphate (AMP), but the third phosphate is not attached by a high-energy bond. With only one phosphate group, AMP has no other phosphates to provide the electrostatic repulsion that makes the bonds holding the two terminal phosphate groups high-energy bonds.

ATP hydrolysis drives endergonic processes Cells use ATP to drive endergonic processes. Remember that endergonic reactions do not proceed spontaneously because their products possess more free energy than their reactants; ΔG for an endergonic reaction is positive. If the ATP hydrolysis releases more energy than the other reaction consumes, then coupling the two reactions will produce an overall ΔG for the coupled reactions that is negative. That is, energy released by the hydrolysis of ATP can supply the energy needed by the endergonic reaction. Because most endergonic reactions in cells require less energy than is released by ATP hydrolysis, ATP can provide most of the energy a cell needs. ATP also powers other cellular functions such as generating force in muscles, or creating concentration gradients of important ions, both processes that require energy.

Data analysis Consider the reaction:

glutamate + NH3 → glutamine (∆G = +3.4 kcal/mol). If this reaction is coupled to ATP hydrolysis (∆G = −7.3 kcal/ mol), what would be the overall ∆G? Would this process be endergonic or exergonic?

stockpiles of ATP. Instead, they typically have only a few seconds’ supply of ATP at any given time, and they continually produce more from ADP and Pi. It is estimated that even a sedentary individual turns over an amount of ATP in one day roughly equal to his or her body weight. This statistic makes clear the importance of ATP synthesis. In chapters 7 and 8 we will explore in detail the cellular mechanisms for synthesizing ATP.

Learning Outcomes Review 6.3

ATP is a nucleotide with three phosphate groups. Endergonic cellular processes can be driven by coupling to the exergonic hydrolysis of the two terminal phosphates. The bonds holding the terminal phosphate groups together are easily broken, releasing energy like a coiled spring. The cell is constantly building ATP using exergonic reactions and breaking it down to drive endergonic reactions. ■■

If the molecular weight of ATP is 507.18 g/mol, and the ∆G for hydrolysis is –7.3 kcal/mol, how much energy is released over the course of the day by a 100-kg man?

6.4 Enzymes:

Catalysts

Biological

Learning Outcomes

ATP cycles continuously The same feature that makes ATP an effective energy donor—the instability of its phosphate bonds—makes it a poor long-term energystorage molecule. Fats and carbohydrates serve that function better. Cells make and use ATP cyclically: cells use exergonic reactions to provide energy to synthesize ATP from ADP + Pi; they then use the hydrolysis of ATP to provide energy to drive endergonic processes (figure 6.7). Most cells do not maintain large

Energy from exergonic cellular reactions

ATP

ADP

+

+

H2O

Energy for endergonic cellular processes

Pi

Figure 6.7 The ATP cycle. ATP is synthesized and hydrolyzed in a cyclic fashion. The synthesis of ATP from ADP + P i is endergonic and is powered by exergonic cellular reactions. The hydrolysis of ATP to ADP + Pi is exergonic, and the energy released is used to power endergonic cellular functions such as muscle contraction. ADP, adenosine diphosphate; ATP, adenosine triphosphate; P i, inorganic phosphate. 118 part II Biology of the Cell

1. Discuss the specificity of enzymes. 2. Explain how enzymes bind to their substrates. 3. List the factors that influence the rate of enzyme-catalyzed reactions.

The chemical reactions within living organisms are regulated by controlling the points at which catalysis takes place. Life itself, therefore, can be seen as regulated by catalysts. This is possible because the catalysts in living systems, enzymes, are also highly specific. Most enzymes are proteins, although RNA molecules can also have catalytic activity.

Enzymes lower the activation energy of reactions The unique three-dimensional shape of an enzyme enables it to stabilize a temporary association between substrates —the molecules that will undergo the reaction. By bringing two substrates together in the correct orientation or by stressing particular chemical bonds of a substrate, an enzyme lowers the activation energy required for new bonds to form. The reaction thus proceeds much more quickly than it would without the enzyme. The enzyme itself is not changed or consumed in the reaction, so only a small amount of an enzyme is needed, and it can be used repeatedly.

As an example of how an enzyme works, let’s consider the reaction of carbon dioxide and water to form carbonic acid. This important enzyme-catalyzed reaction occurs in vertebrate red blood cells: CO2 + H2O ⇋ H2CO3 carbon water carbonic dioxide acid This reaction may proceed in either direction, but because it has a large activation energy, the reaction is very slow in the absence of an enzyme: perhaps 200 molecules of carbonic acid form in an hour in a cell in the absence of any enzyme. Reactions that proceed this slowly are of little use to a cell. Vertebrate red blood cells overcome this problem by employing an enzyme within their cytoplasm called carbonic anhydrase (enzyme names usually end in “–ase”). Under the same conditions, but in the presence of carbonic anhydrase, an estimated 600,000 molecules of carbonic acid form every second! Thus, the enzyme increases the reaction rate by more than one million times. Thousands of different kinds of enzymes are known, each catalyzing one or a few specific chemical reactions. By facilitating particular chemical reactions, the enzymes in a cell determine the course of metabolism—the collection of all chemical reactions— in that cell. Different types of cells contain different sets of enzymes, and this difference contributes to structural and functional variations among cell types. For example, the chemical reactions taking place within a red blood cell differ from those that occur within a nerve cell, in part because different cell types contain different arrays of enzymes.

Active sites of enzymes conform to fit the shape of substrates Most enzymes are globular proteins with one or more pockets or clefts, called active sites, on their surface (figure 6.8). Substrates

Active site

Substrate

Enzyme

a.

Enzyme–substrate complex

b.

Figure 6.8 Enzyme binding its substrate. a. The active

site of the enzyme lysozyme fits the shape of its substrate, a peptidoglycan that makes up bacterial cell walls. b. When the substrate, indicated in yellow, slides into the groove of the active site, the protein is induced to alter its shape slightly and bind the substrate more tightly. This alteration of the shape of the enzyme to better fit the substrate is called induced fit.

SCIENTIFIC THINKING Hypothesis: Protein structure is flexible, not rigid. Prediction: Antibody–antigen binding can involve a change in protein structure. Test: Determine crystal structure of a fragment of a specific antibody with no antigen bound, and with antigen bound for comparison. No antigen

Bound antigen

Result: After binding, the antibody folds around the antigen, forming a pocket. Conclusion: In this case, binding involves an induced-fit kind of change in conformation. Further Experiments: Why is this experiment easier to do with an antibody than with an enzyme? Can this experiment be done with an enzyme?

Figure 6.9 Induced-fit binding of antibody to antigen.

bind to the enzyme at these active sites, forming an enzyme–­ substrate complex (see figure 6.10). For catalysis to occur within the complex, a substrate molecule must fit precisely into an active site. When that happens, amino acid side groups of the enzyme end up very close to certain bonds of the substrate. These side groups interact chemically with the substrate, usually stressing or distorting a particular bond and consequently lowering the activation energy needed to break the bond. After the bonds of the substrates are broken, or new bonds are formed, the substrates have been converted to products. These products then dissociate from the enzyme, leaving the enzyme ready to bind its next substrate and begin the cycle again. Proteins are not rigid. The binding of a substrate induces the enzyme to adjust its shape slightly, leading to a better induced fit between enzyme and substrate (figure 6.9). This interaction may also facilitate the binding of other substrates; in such cases, one substrate “activates” the enzyme to receive other substrates.

Enzymes occur in many forms Although many enzymes are suspended in the cytoplasm of cells, not attached to any structure, other enzymes function as integral parts of cell membranes and organelles. Enzymes may also form associations called multienzyme complexes to carry out reaction sequences. And it is now clear that some enzymes may contain catalytic RNA rather than being only protein. chapter

6 Energy and Metabolism 119

3. The binding of the substrate and enzyme places stress on the glucose– fructose bond, and the bond breaks.

1. The substrate, sucrose, consists of glucose and fructose bonded together. 2. The substrate binds to the active site of the enzyme, forming an enzyme– substrate complex.

Bond

H2O

Glucose Fructose

4. Products are released, and the enzyme is free to bind other substrates.

Active site Enzyme sucrase

Figure 6.10 The catalytic cycle of an enzyme. Enzymes increase the speed at which chemical reactions occur, but they are not altered permanently themselves as they do so. In the reaction illustrated here, the enzyme sucrase is splitting the sugar sucrose into two simpler sugars: glucose and fructose.

Multienzyme complexes Often several enzymes catalyzing different steps of a sequence of ­reactions are associated with one another in noncovalently ­bonded assemblies called multienzyme complexes. The bacterial ­pyruvate dehydrogenase multienzyme complex, shown in figure 6.11, contains enzymes that carry out three sequential reactions in oxidative metabolism. Each complex has multiple copies of each of the three enzymes—60 protein subunits in all. The a. many subunits work together to form a molecular machine that ­performs multiple functions. Multienzyme complexes offer the following significant advantages in catalytic efficiency: 1. The rate of any enzyme reaction is limited by how often the enzyme collides with its substrate. If a series of sequential reactions occurs within a multienzyme complex, the product of one reaction can be delivered to the next enzyme without releasing it to diffuse away. 2. Because the reacting substrate doesn’t leave the complex while it goes through the series of reactions, unwanted side reactions are prevented. 3. All of the reactions that take place within the multienzyme complex can be controlled as a unit. In addition to pyruvate dehydrogenase, which controls entry to the citric acid cycle during aerobic respiration (see chapter 7), several other key processes in the cell are catalyzed by ­multienzyme complexes. One well-studied system is the fatty acid synthetase 120 part II Biology of the Cell

b.

50 nm

Figure 6.11 A complex enzyme: pyruvate dehydrogenase. Pyruvate dehydrogenase, which catalyzes the

oxidation of pyruvate, is one of the most complex enzymes known. a. A model of the enzyme showing the arrangement of the 60 protein subunits. b. Many of the protein subunits are clearly visible in the electron micrograph.  (b) Professor Emeritus Lester J. Reed, University of Texas

at Austin

The original enzymes discovered and studied were all proteins. For many years, we assumed that all enzymes were proteins until Thomas R. Cech and colleagues at the University of Colorado reported in 1981 an RNA splicing reaction that did not require protein. Around the same time, Sidney Altman and Norman Pace were studying the enzyme RNase P, and found that this enzyme was composed of both protein and RNA, and further, that the RNA was the catalytic molecule. Like protein enzymes, these RNA catalysts, which are called ­“ribozymes,” greatly accelerate the rate of particular biochemical reactions and show extraordinary substrate specificity. Research has revealed at least two sorts of ribozymes. Some ribozymes have folded structures and catalyze reactions on themselves, a process called intramolecular catalysis. Other ribozymes act on other molecules without being changed themselves, a process called intermolecular catalysis. Perhaps the most striking example of enzymatic RNA is in the structure and function of the ribosome, the protein synthetic machine composed of RNA and protein. For many years, it was assumed that RNA provided a structural framework to facilitate the enzymatic activity of ribosomal proteins. It is now apparent that ribosomal proteins form the structural scaffold that promotes the enzymatic activity of ribosomal RNA. The ability of RNA, an informational molecule, to act as a catalyst has stirred great excitement because it seems to address the question, “Which came first, the protein or the nucleic acid?” It now appears likely that RNA evolved first and may have catalyzed the formation of the first proteins.

Enzyme function is sensitive to environmental factors The rate of an enzyme-catalyzed reaction is affected by the concentrations of both the substrate and the enzyme that works on it. In addition, any chemical or physical factor that alters the enzyme’s three-dimensional shape—such as temperature, pH, and the binding of regulatory molecules—can affect the enzyme’s ability to catalyze the reaction.

Temperature Increasing the temperature of an uncatalyzed reaction increases its rate because the additional heat increases random molecular movement. This increases the fraction of molecules whose kinetic ­energy is greater than the activation energy for the reaction. The rate of an enzyme-catalyzed reaction also increases with temperature, but only up to a point called the optimum temperature (figure 6.12a). Below this temperature, the hydrogen bonds and hydrophobic interactions that determine the enzyme’s shape are not flexible enough to permit the induced fit that is optimum for catalysis. Above the optimum temperature, these forces are too weak to maintain the enzyme’s shape against the increased random

Rate of Reaction

Nonprotein enzymes

Optimum temperature for human enzyme

30

40

50

Optimum temperature for enzyme from hot springs prokaryote

60

70

80

Temperature of Reaction (˚C)

a. Rate of Reaction

complex that catalyzes the synthesis of fatty a­ cids from two-carbon precursors. Seven different enzymes make up this multienzyme complex, and the intermediate reaction products remain associated with the complex for the entire series of reactions.

Optimum pH for pepsin

1

2

3

Optimum pH for trypsin

4 5 6 pH of Reaction

7

8

9

b.

Figure 6.12 Enzyme sensitivity to the environment. 

The activity of an enzyme is influenced by both (a) temperature and (b) pH. Most human enzymes, such as the protein-degrading enzyme trypsin, work best at temperatures of about 40°C and within a pH range of 6 to 8. The hot springs prokaryote tolerates a higher environmental temperature and a correspondingly higher temperature optimum for enzymes. Pepsin works in the acidic environment of the stomach and has a lower optimum pH.

movement of the atoms in the enzyme. At higher temperatures, the enzyme denatures, as described in chapter 3. Most human enzymes have an optimum temperature between 35°C and 40°C—a range that includes normal body temperature. Prokaryotes that live in hot springs have more stable enzymes (that is, enzymes held together more strongly), so the optimum temperature for those enzymes can be 70°C or higher. In each case the optimal temperature for the enzyme corresponds to the “normal” temperature usually encountered in the body or the environment, depending on the type of organism.

pH Ionic interactions between oppositely charged amino acid residues, such as glutamic acid (−) and lysine (+), also hold enzymes together. These interactions are sensitive to the hydrogen ion concentration of the fluid in which the enzyme is dissolved, because changing that concentration shifts the balance between positively and negatively charged amino acid residues. For this reason, most enzymes have an optimum pH that usually ranges from pH 6 to 8. Enzymes able to function in very acidic environments are proteins that maintain their three-dimensional shape even in the presence of high hydrogen ion concentrations. The enzyme pepsin, for example, digests proteins in the stomach at pH 2, a very acidic level (figure 6.12b).

Inhibitors and activators Enzyme activity is also sensitive to the presence of specific substances that can bind to the enzyme and cause changes in its chapter

6 Energy and Metabolism 121

shape. Through these substances, a cell is able to regulate which of its enzymes are active and which are inactive at a particular time. This ability allows the cell to increase its efficiency and to control changes in its characteristics during development. A substance that binds to an enzyme and decreases its activity is called an inhibitor. Very often, the end product of a biochemical pathway acts as an inhibitor of an early reaction in the p­ athway, a process called feedback inhibition (discussed in ­section 6.5). Enzyme inhibition occurs in two ways: competitive inhibitors compete with the substrate for the same active site, occupying the active site and thus preventing substrates from binding; noncompetitive inhibitors bind to the enzyme in a location other than the active site, changing the shape of the enzyme and making it unable to bind to the substrate (figure 6.13). Many enzymes can exist in either an active or an inactive conformation; such enzymes are called allosteric enzymes. Most noncompetitive inhibitors bind to a specific portion of the enzyme called an allosteric site. These sites serve as chemical on/off switches; the binding of a substance to the site can switch the enzyme between its active and inactive configurations. A substance that binds to an allosteric site and reduces enzyme activity is called an allosteric inhibitor (figure 6.13b). This kind of control is also used to activate enzymes. An allosteric activator binds to allosteric sites to keep an enzyme in its active configuration, thereby increasing enzyme activity.

When the cofactor is a nonprotein organic molecule, it is called a coenzyme. Many of the small organic molecules essential in our diets that we call vitamins function as coenzymes. For example, the B vitamins B6 and B12 both function as coenzymes for a number of ­different enzymes. Modified nucleotides are also used as coenzymes. In numerous oxidation–reduction reactions that are catalyzed by enzymes, the electrons pass in pairs from the active site of the enzyme to a coenzyme that serves as the electron acceptor. The coenzyme then transfers the electrons to a different enzyme, which releases them (and the energy they bear) to the substrates in another reaction. Often, the electrons combine with protons (H+) to form hydrogen atoms. In this way, coenzymes shuttle energy in the form of hydrogen atoms from one enzyme to another in a cell. The role of coenzymes and the specifics of their action will be explored in detail in chapters 7 and 8.

Learning Outcomes Review 6.4

Enzymes are biological catalysts that accelerate chemical reactions inside the cell. Enzymes bind to their substrates based on molecular shape, which allows them to be highly specific. Enzyme activity is affected by conditions such as temperature and pH and the presence of inhibitors or activators. Some enzymes also require an inorganic cofactor or an organic coenzyme. ■■

Enzyme cofactors Enzyme function is often assisted by additional chemical components known as cofactors. These can be metal ions that are often found in the active site participating directly in catalysis. For example, the metallic ion zinc is used by some enzymes, such as protein-digesting carboxypeptidase, to draw electrons away from their position in covalent bonds, making the bonds less stable and easier to break. Other metallic elements, such as molybdenum and manganese, are also used as cofactors. Like zinc, these substances are required in the diet in small amounts.

Why do proteins and RNA function as enzymes whereas DNA does not?

6.5 Metabolism:

The Chemical Description of Cell Function

Learning Outcomes

Active site

1. Explain the kinds of reactions that make up metabolism. 2. Discuss what is meant by a metabolic pathway. 3. Recognize that metabolism is a product of evolution.

Substrate

Substrate

Inhibitor

Enzyme

Active site

Inhibitor

Enzyme Allosteric site

Competitive inhibitor interferes with active site of enzyme so substrate cannot bind

a. Competitive inhibition

Allosteric inhibitor changes shape of enzyme so it cannot bind to substrate

b. Noncompetitive inhibition

Figure 6.13 How enzymes can be inhibited. a. In competitive inhibition, the inhibitor has a shape similar to the substrate and competes for the active site of the enzyme. b. In noncompetitive inhibition, the inhibitor binds to the enzyme at the allosteric site, a place away from the active site, effecting a conformational change in the enzyme, making it unable to bind to its substrate. 122 part II Biology of the Cell

Living chemistry, the total of all chemical reactions carried out by an organism, is called metabolism. Those chemical reactions that expend energy to build up molecules are called anabolic reactions, or anabolism. Reactions that harvest energy by breaking down molecules are called catabolic reactions, or catabolism. We have already seen examples of this in chapter 3, where we saw that ­biopolymers are built (anabolism) by d­ ehydration reactions, and degraded (catabolism) by hydrolysis reactions.

Biochemical pathways organize chemical reactions in cells Organisms contain thousands of different kinds of enzymes that catalyze a bewildering variety of reactions. Many of these reactions

in a cell occur in sequences called biochemical pathways. In such pathways, the product of one reaction becomes the substrate for the next (figure 6.14). Biochemical pathways are the organizational units of metabolism—the elements an organism controls to achieve coherent metabolic activity. Many sequential enzyme steps in biochemical pathways take place in specific compartments of the cell; for example, the steps of the citric acid cycle (see chapter 7) occur in the matrix inside mitochondria in eukaryotes. By determining where many of the enzymes that catalyze these steps are located, we can “map out” a model of metabolic processes in the cell. The majority of these biochemical pathways are common to all cells, which implies that basic cellular metabolism evolved early in the history of life on this planet. This has left modern cells with a metabolism where carbon and energy pathways converge to produce 12 key intermediates that are also the precursors for biosynthetic pathways.

Feedback inhibition regulates some biochemical pathways

Initial substrate

Enzyme 2

Enzyme 3

a.

Enzyme 1 Intermediate substrate A Enzyme 2 Intermediate substrate B Enzyme 3 Intermediate substrate C Enzyme 4

For a cell to operate efficiently, the biochemical pathways that make ups its metabolism must be coordinated. Not only is it unnecessary to synthesize a compound when enough is available, but it is a waste of energy and raw materials that could be used elsewhere in the cell. It is advantageous to the cell for biochemical pathways to be active only when their products are needed. The regulation of simple biochemical pathways often depends on an elegant feedback mechanism: the end-product of the pathway binds to an allosteric site on the enzyme that catalyzes the first reaction in the pathway. This mode of regulation is called feedback inhibition. Consider a hypothetical biochemical pathway with 3 enzymes, and initial substrate that is converted into the end-product via two intermediate substrates A and B (figure 6.15). With no feedback inhibition, the pathway keeps churning out end product as

Enzyme 1

Initial substrate

End product

Figure 6.14 A biochemical pathway.  The original

substrate is acted on by enzyme 1, changing the substrate to a new intermediate, substrate A, recognized as a substrate by enzyme 2. Each enzyme in the pathway acts on the product of the previous stage. These enzymes may be either soluble or arranged in a membrane as shown.

Initial substrate

Enzyme 1

Intermediate substrate A

Enzyme 2

Intermediate substrate B

End-product

Enzyme 3

Figure 6.15 Feedback inhibition. a. A biochemical

pathway with no feedback inhibi­tion. b. A biochemical pathway in which the final end-product becomes the allosteric inhibitor for the first enzyme in the pathway. In other words, the formation of the pathway’s final end-product stops the pathway. The pathway could be the synthesis of an amino acid, a nucleotide, or another important cellular molecule.

End-product

b. chapter

6 Energy and Metabolism 123

long as the initial substrate is available. With feedback inhibition, as end product builds up, it can bind to an allosteric site in enzyme 1 to shut off the pathway. In this chapter we have reviewed the basics of energy and its transformations as carried out in living systems. Chemical bonds are the primary location of energy storage and release, and cells have developed elegant methods of making and breaking chemical bonds to create the molecules they need. Enzymes facilitate these reactions by serving as catalysts. In chapters 7 and 8 you will learn the details of the mechanisms by which organisms harvest, store, and utilize energy.

Learning Outcomes Review 6.5

Metabolism is the sum of all chemical reactions in a cell. Anabolic reactions use energy to build up molecules. Catabolic reactions release energy by breaking down molecules. In a metabolic pathway, the end-product of one reaction is the substrate for the next reaction. Evolution may have favored organisms that could use precursor molecules to synthesize a nutrient. Over time, more reactions would be linked together as novel enzymes arose by mutation. ■■

Is a catabolic pathway likely to be subject to feedback inhibition?

Chapter Review Robert Caputo/Aurora Photos/Cavan images

6.1

The Flow of Energy in Living Systems

Thermodynamics is the study of energy changes.

with a positive ΔG are not spontaneous (endergonic). Endergonic chemical reactions absorb energy from the surroundings, whereas exergonic reactions release energy to the surroundings.

Energy can take many forms. Energy is the capacity to do work. Potential energy is stored energy, and kinetic energy is the energy of motion. Energy can take many forms: mechanical, heat, sound, electric current, light, or radioactive radiation. Energy is measured in units of heat known as kilocalories.

Spontaneous chemical reactions require activation energy. Activation energy is the energy required to destabilize chemical bonds and initiate chemical reactions (figure 6.5). Even exergonic reactions require this activation energy. Catalysts speed up chemical reactions by lowering the activation energy.

The Sun provides energy for most living systems. Photosynthesis stores light energy from the Sun as potential energy in the covalent bonds of sugar molecules. Breaking these bonds in living cells releases energy for use in other reactions.

6.3 ATP: The Energy Currency of Cells

Redox reactions transfer electrons. Oxidation is a reaction involving the loss of electrons. Reduction is the gain of electrons (figure 6.2). These two reactions take place together and are therefore termed redox reactions.

6.2 The Laws of Thermodynamics and Free Energy The First Law states that energy cannot be created or destroyed. Virtually all activities of living organisms require energy. Energy changes form as it moves through organisms and their biochemical systems, but it is not created or destroyed. The Second Law states that some energy is lost as disorder increases. The disorder, or entropy, of the universe is continuously increasing. In an open system like the Earth, which is receiving energy from the Sun, this may not be the case. To increase order, however, energy must be expended. In energy conversions, some energy is always lost as heat. Chemical reactions can be predicted based on changes in free energy. Free energy (G) is the energy available to do work in any system. Changes in free energy (ΔG) predict the direction of reactions. Reactions with a negative ΔG are spontaneous (exergonic) reactions, and reactions 124 part II Biology of the Cell

Adenosine triphosphate (ATP) is the molecular currency used for cellular energy transactions. Cells store and release energy in the bonds of ATP. The energy of ATP is stored in the bonds between its terminal phosphate groups. These groups repel each other due to their negative charge and therefore the covalent bonds joining these phosphates are unstable. ATP hydrolysis drives endergonic processes. Enzymes hydrolyze the terminal phosphate group of ATP to release energy for reactions. If ATP hydrolysis is coupled to an endergonic reaction with a positive ΔG with magnitude less than that for ATP hydrolysis, the two reactions together will be exergonic. ATP cycles continuously. ATP hydrolysis releases energy to drive endergonic reactions, and it is synthesized with energy from exergonic reactions (figure 6.7).

6.4 Enzymes: Biological Catalysts Enzymes lower the activation energy of reactions. Enzymes lower the activation energy needed to initiate a chemical reaction. Active sites of enzymes conform to fit the shape of substrates. Substrates bind to the active site of an enzyme. Enzymes adjust their shape to the substrate so there is a better fit (figure 6.8). Enzymes occur in many forms. Enzymes can be free in the cytosol or exist as components bound to membranes and organelles. Enzymes involved in a biochemical

6.5  Metabolism: The Chemical Description

pathway can form multienzyme complexes. Although most enzymes are proteins, some are actually RNA molecules, called ribozymes.

of Cell Function

Metabolism is the sum of all biochemical reactions in a cell. Anabolic reactions require energy to build up molecules, and catabolic reactions break down molecules and release energy.

Enzyme function is sensitive to environmental factors. An enzyme’s functionality depends on its ability to maintain its threedimensional shape, which can be affected by temperature and pH. The activity of enzymes can be affected by inhibitors. Competitive inhibitors compete for the enzyme’s active site, which leads to decreased enzyme activity (figure 6.13). Enzyme activity can be controlled by effectors. Allosteric enzymes have a second site, located away from the active site, that binds effectors to activate or inhibit the enzyme. Noncompetitive inhibitors and activators bind to the allosteric site, changing the structure of the enzyme to inhibit or activate it. Cofactors are nonorganic metals necessary for enzyme function. Coenzymes are nonprotein organic molecules, such as certain vitamins, needed for enzyme function. Often coenzymes serve as electron acceptors.

Biochemical pathways organize chemical reactions in cells. Chemical reactions in biochemical pathways use the product of one reaction as the substrate for the next. Carbon and energy pathways converge to produce 12 key intermediates that are also the precursors for biosynthetic pathways. Feedback inhibition regulates some biochemical pathways. Biosynthetic pathways are often regulated by the end-product of the pathway. Feedback inhibition occurs when the end-product of a reaction combines with an enzyme’s allosteric site to shut down the enzyme’s activity (figure 6.15).

Visual Summary transferred in

Energy

Metabolism

in cells

requires Chemical reactions

ATP requires energy

based on Changes in free energy

Enzymes

Cellular chemical reactions

are

types

releases energy

or

ΔG>0 Require energy

Spontaneous

Not spontaneous

Specificity

Use energy Build molecules

lower

Release energy Catabolic Break down molecules are affected by

bind

with

Biochemical pathways

Biological catalysts

Activation energy

Endergonic reactions

ΔG0.5%. These have been identified, and mapped by an international consortium, with the data being publicly available.

Genetic maps can be used to find alleles that cause genetic disorders With these common molecular markers in hand, a dense genetic map was constructed as part of the Human Genome Project (see chapter 18). This genetic map provides a framework to locate genes of interest, that is, those that affect interesting phenotypes. Human genes can be mapped, similar to genetic mapping in model organisms, but using data derived from families instead of controlled crosses. The principle is the same—genetic distance is still proportional to recombination frequency—but the analysis requires the use of complex statistics and summing data from many families. In addition to genetic maps, there are a variety of physical maps that can be constructed as well. This combination of genetic and physical mapping was an integral part of the Human Genome Project. Here we will consider the use of genetic mapping and of association studies in human genetics. The power of genetic mapping is that it allows geneticists to find the genes responsible for specific phenotypes. Even before the Human Genome Project was completed, geneticists were finding genes that could be mutated to cause particular genetic diseases, including cystic fibrosis (recessive allele) and Huntington disease (dominant allele). Linkage analysis can identify rare causal variants in a population that have a large effect on phenotype. This limits gene discovery using linkage analysis to genetic disorders caused by a single gene, so-called monogenic disorders. The logic is to follow the segregation of possible causal alleles with disease state in multiple families. The more data available, the smaller region of the genome this can identify. While the process is laborious, and time consuming, by the time of the publication of the sequence of the human genome (see chapter 18), around 1,000 single-gene inherited diseases had been characterized. Where linkage-based analysis is not effective is in determining the genes involved in complex traits affected by many genes. The advent of the data from the Human Genome Project led to the possibility of analyzing complex traits with no single variant with a large effect.

Genome-wide association studies (GWAS) can find rare causal variants Although there are estimated to be some 7000 inherited diseases caused by rare variants of single genes, most human phenotypes, including many common diseases, are affected by many genes. We saw in the last chapter that complex traits are usually so-called

quantitative traits that are due to the action of many genes. To address these traits, a different methodology was needed, although still a phenotype-to-genotype, forward genetics approach. Thus the genome-wide association study, or GWAS, was born in 2005. In these studies, it is possible to start with a complex disease phenotype, for example, coronary artery disease, then look for SNPs associated with this phenotype in a population. As the location of each SNP is known, you can then look for candidate genes in that region of the genome. Coronary artery disease is an interesting example, as a few variants were identified with relatively large effects. One variant found in both U.S. and European populations is the most common genetic risk factor and accounts for more than 10% of disease risk. But this and other variants with large effects do not account for a majority of the heritability. There are literally thousands of variants with small effects that contribute to coronary artery disease, and account for the majority of the heritability. This pattern has held true for most complex diseases analyzed by GWAS. GWAS have also been used to look at complex traits that have high heritability (see chapter 12) such as height and body weight. For height, over 700 common variants (a frequency of over 5% in the population) have been shown to affect height, each with a relatively small effect. A smaller number of rare variants have also been identified, but detection requires analyzing very large populations. Most GWAS to date have used techniques that involve analyzing SNPs, and thus are limited by the number of SNPs assayed. The more rare variants that are used, the more that are found to have small effects. At this point, while they have provided a mountain of data (over 3000 studies), GWAS are still a young technology. Despite this, they are changing how we view human phenotypes. Complex human phenotypes appear to be affected by thousands of relatively common genetic variants, each with small individual effect, which are predominantly found in noncoding regions. These most likely affect gene expression, and thus a single variant can affect multiple phenotypes. Rare variants affect human phenotypes as well, but their identification is hampered by the size of populations required to provide sufficient statistical power. It is important to emphasize that finding a variant that is associated with a phenotype does not mean the variant itself is causal. The variant may be close to a gene that affects a phenotype, but the variant itself does not. Or, the variant may affect the regulation of a gene with a direct effect. The greatest limitation to these studies is that they tell us nothing about the function of the variants identified.

Reverse genetics are now possible in humans Both linkage analysis and GWAS use the logic of forward genetics, that is, going from a known phenotype to an unknown genotype (figure 13.18). The only mammalian system that has powerful techniques of reverse genetics is mouse (see chapter 17). The ability to knock out genes in the mouse is a powerful use of reverse genetics to discover the function of specific genes. While this is not possible in humans, we can look for potential loss-of-function (LOF) alleles, such as ones that result in drastically shortened proteins, which are expected to lack function. This work is still in the early stages, but putative LOF variants for thousands of genes are being identified. These are mainly at low chapter

13 The Chromosomal Basis of Inheritance, and Human Genetics 263

Forward genetics

Linkage analysis - Uses family data - Single variants with large effects GWAS - Uses population data - Many variants with small effects Environment

Reverse genetics

Genotype

Phenotype

PheWAS - Uses population data - Requires EHR and genetic data

Figure 13.18 Analyze genotype–phenotype relationships. We can study the relationship between genes and

phenotypes by using traditional forward genetics, where we start with a specific phenotype and try to find the causal genes. In humans this involves linkage analysis and GWAS. Reverse genetics inactivates genes, then analyzes the resulting phenotype. In humans, we can do this using PheWAS, looking for associations of genetic variants to phenotypes in electronic health records (EHR).

frequency (a frequency of 0.1% or less) in the population, so the number that are homozygous is extremely low, but these represent genes that can be considered nonessential, although of still unknown function. Despite the early state of these data, around 1800 genes have been identified as having putative LOF variants that can exist as homozygotes. The next step in this approach is to correlate homozygous LOF variants with possible phenotypes. At least one group is moving towards a Human Knockout Gene Project.

Another approach is to take advantage of large databases containing detailed phenotypic data linked to genotypic data. Some electronic health records (EHR) are being designed to standardize phenotypic data, and when these include volunteers who provide DNA for analysis, this allows a form of reverse genetics in humans. This kind of population-based analysis, the phenotypewide association study (PheWAS), uses known variants to assess their association with a wide spectrum of phenotypes in the EHR. These PheWAS can reveal the function of unknown variants, and begin to unravel how many variants affect different phenotypes. This can also be used to identify variants that affect more than one phenotype, or pleiotropy (see chapter 12). Early results indicate that many common variants affect multiple phenotypes, implying high levels of pleiotropy. The next step is to unify the approaches of GWAS and PheWAS to explore both sides of the genotype-phenotype relationship.

Learning Outcomes Review 13.5

Linkage analysis can be used to find the genes responsible for specific phenotypes. This will find variants with large effects, usually monogenic disorders. GWAS use data from populations to find associations between a specific phenotype and many variants of small effect. Both of these are forward genetics going from phenotype to genotype. Reverse genetics in humans uses PheWAS, where specific genetic variants can be used to look for associations to phenotypes in electronic health data. ■■

Would you expect a GWAS kind of approach to find the basis of a single-gene disorder?

Chapter Review Adrian T Sumner/Science Source

13.1 Sex Linkage and the Chromosomal Theory

In some animals, females have two similar sex chromosomes and males have sex chromosomes that differ. In other species, females have sex chromosomes that differ (table 13.1).

Morgan correlated the inheritance of a trait with sex chromosomes (figure 13.2). Morgan crossed red-eyed and white-eyed flies and found differences in inheritance based on the sex of offspring. All white-eyed offspring were males, but testcrosses showed that white-eyed females were possible, supporting the idea that the white-eye gene was on the X chromosome.

Dosage compensation prevents doubling of sex-linked gene products. In fruit flies, males double the gene expression from their single X chromosome. In mammals, one of the X chromosomes in a female is randomly inactivated during development. In a mammalian female that is heterozygous for X-chromosome alleles, X inactivation produces a mosaic pattern, as shown in the coat color of calico cats (figure 13.3).

of Inheritance

The gene for eye color lies on the X chromosome. The inheritance of eye color in Drosophila segregates with the X chromosome, a phenomenon termed sex-linked inheritance. In humans, the Y chromosome generally determines maleness. The Y chromosome is highly condensed and does not have active counterparts to most genes on the X chromosome. Presence of a Y chromosome leads to development of male gonads. This is called chromosomal sex determination. 264 part III Genetic and Molecular Biology

13.2 Exceptions to Mendelian Inheritance Mitochondrial genes are inherited from the female parent. Mitochondria have their own genomes and divide independently; they are passed to offspring in the cytoplasm of the egg cell.

Chloroplast genes may also be passed on uniparentally. Chloroplasts also reside in the cytoplasm, have their own genomes, and divide independently. They are usually inherited maternally. Genomic imprinting depends on the parental origin of alleles. In genomic imprinting, the expression of a gene depends on parent of origin. This is an example of epigenetics, where a heritable change in phenotype is not due to a change to DNA. Imprinting produces a haploid phenotype.

13.3 Genetic Mapping Mendel’s independent assortment is too simplistic. Genes on the same chromosome may or may not segregate independently. Genetic recombination exchanges alleles on homologues. Homologous chromosomes may exchange alleles by crossing over (figure 13.5). This occurs by breakage and rejoining of chromosomes, as shown by crosses in which chromosomes carry both visible and genetic markers (figure 13.6). Recombination is the basis for genetic maps. Genes close together on a single chromosome are said to be linked. The farther apart two linked genes are, the greater the frequency of recombination. Genetic maps are constructed based on recombination frequency. 1 map unit is 1% recombinant progeny. Multiple crossovers can yield independent assortment results. The probability of multiple crossovers increases with distance between genes, and results in an underestimate of recombination frequency. Maximum recombination frequency is 50%, or independent assortment. Three-point crosses can be used to put genes in order (figure 13.9). If three genes are used, data from multiple crossovers can be used to order genes. Longer map distances are underestimates of true distance due to double crossovers. Evaluating intervening genes with less separation leads to more accurate distances.

13.4 Human Genetics Some human traits exhibit simple dominant/recessive inheritance. Human traits have been observed to follow both simple dominant and recessive inheritance. Human pedigrees symbolize relationships. We analyze human inheritance using pedigrees, which show familial relationships with males as squares and females as circles. Human genetic disorders can be sex linked. Recessive traits on the X chromosome will be visible in males, and are inherited from the mother. Alterations to hemoglobin genes lead to anemia. The phenotypes in sickle cell anemia can all be traced to alterations in the structure of hemoglobin that affect the shape of red blood cells. Over

700 variants of hemoglobin structure have been characterized, some of which also cause disorders. How many human genes can lead to Mendelian phenotypes?. Experiments in the mouse imply that mutations in a majority of human genes would produce a phenotype. Online databases like OMIM catalog human genetic diseases. There are currently 6771 single-gene disorders known. Nondisjunction of chromosomes changes chromosome number. Nondisjunction or failure of separation during meiosis results in aneuploidy: monosomy or trisomy of a chromosome in the zygote. Most are lethal, but some (trisomy 21 in humans), can result in viable offspring. Sex chromosome nondisjunction produces XX, YY, or gametes with no sex chromosomes (figure 13.15). Nondisjunction is more common on the female’s side, and the frequency increases with age. Some genetic defects can be detected early in pregnancy. Genetic defects in humans can be determined by pedigree analysis, amniocentesis, chorionic villus sampling, or noninvasive prenatal testing.

13.5 Human Genetic Mapping and Association Studies Traditional forward genetics goes from phenotype to genotype. Reverse genetics goes from genotype to phenotype. Studying human genetics this way requires using family data, and the large number of genetic variants known. The difficulty of mapping in humans. Genetic mapping in humans was slow to develop because it required multiple disease-causing alleles segregating in a family. The development of molecular marker with no phenotype allowed construction of a detailed map. Genetic maps can be used to find alleles that cause genetic disorders. This is done by following segregation of disease alleles in multiple families. More than 1000 genes have been identified in this way. This can identify monogenic disorders. Genome-wide association studies (GWAS) can find rare causal variants. Linkage mapping is not useful with complex phenotypes caused by quantitative traits. Genome-wide association studies allow the analysis of associations of phenotype with many possible genetic variants. Complex loci are affected by large numbers of small- effect variants. Reverse genetics are now possible in humans. The first attempts at reverse genetics in humans looked at loss-offunction alleles. Homozygous LOF alleles must be nonessential. PheWAS allow searching for associations between specific variants and many phenotypes in electronic health records.

chapter

13 The Chromosomal Basis of Inheritance, and Human Genetics 265

Visual Summary Exceptions

Chromosomal theory of inheritance

Pedigrees

uses

Human genetics

to

include Genetic mapping

Sex chromosomes Genomic imprinting

require

uses

Map genes

Gene linkage

uses

include

to study

GWAS

Genetic disorders

type of

PheWAS

caused by

Epigenetic inheritance

lead to

Dosage compensation

Genetic recombination

due to

achieved by Organelle inheritance

Association studies

uses

Sex linkage on X chromosome

X-inactivation

Recombination frequency

Mitochondria

results in

measure of

Chloroplasts

Genetic mosaics

Crossing over

Gene mutations

Sickle-cell anemia

Nondisjunction

Down syndrome

Dominant

Juvenile onset glaucoma

Recessive

Albinism

X-linked

Hemophilia

types

Map distance

example Calico cats

Review Questions Adrian T Sumner/Science Source

U N D E R S TA N D 1. Why is the white-eye phenotype always observed in males carrying the white-eye allele? a. b. c. d.

Because the trait is dominant Because the trait is recessive Because the allele is located on the X chromosome and males have only one X Because the allele is located on the Y chromosome and only males have Y chromosomes

2. In an organism’s genome, autosomes are a. b. c. d.

the chromosomes that differ between the sexes. chromosomes that are involved in sex determination. inherited only from the mother (maternal inheritance). all of the chromosomes other than sex chromosomes.

3. What cellular process is responsible for genetic recombination? a.

The independent alignment of homologous pairs during meiosis I

266 part III Genetic and Molecular Biology

b. c. d.

Separation of the homologues in meiosis I Separation of the chromatids during meiosis II Crossing over between homologues

4. Genetic mapping and GWAS a. b. c. d.

are examples of forward genetics. are examples of reverse genetics. both use population data. both rely on family data.

5. How many map units separate two alleles if the recombination frequency is 0.07? a.

700 cM

b.

70 cM

c.

7 cM

d.

0.7 cM

6. How does maternal inheritance of mitochondrial genes differ from sex linkage? a. b.

Mitochondrial genes do not contribute to the phenotype of an individual. Because mitochondria are inherited from the mother, only females are affected.

c. d.

Since mitochondria are inherited from the mother, females and males are equally affected. Mitochondrial genes must be dominant. Sex-linked traits are typically recessive.

7. Which of the following genotypes due to nondisjunction of sex chromosomes is lethal? a.

XXX

b.

XXY

c.

OY

d.

XO

A P P LY 1. A recessive sex-linked gene in humans leads to a loss of sweat glands. A woman heterozygous for this will a. b. c. d.

have no sweat glands. have normal sweat glands. have patches of skin with and without sweat glands. have an excess of sweat glands.

2. Bipolar disorder is a complex trait that shows high heritability in twin studies. The best way to analyze the genetics of this would be to use a. b. c. d.

genetic mapping and family studies to find the gene responsible for bipolar disorder. GWAS to look for associations of genetic variants with bipolar disorder. the twin data to directly map the gene. reverse genetics to find the gene.

3. Down syndrome is the result of trisomy for chromosome 21. Why is this trisomy viable whereas trisomy for most other chromosomes is not? a. b. c. d.

Chromosome 21 is a large chromosome and excess genetic material is less harmful. Chromosome 21 behaves differently in meiosis I than the other chromosomes. Chromosome 21 is a small chromosome with few genes so this does less to disrupt the genome. Chromosome 21 is less prone to nondisjunction than other chromosomes.

4. Genes that are on the same chromosome can show independent assortment a. b. c. d.

5. The A and B genes are 10 cM apart on a chromosome. If an A B/a b heterozygote is testcrossed to a b/a b, how many of each progeny class would you expect out of 100 total progeny? a. b. c. d.

25 A B, 25 a b, 25 A b, 25 a B 10 A B, 10 a b 45 A B, 45 a b 45 A B, 45 a b, 5 A b, 5 a B

6. During the process of spermatogenesis, a nondisjunction event that occurs during the second division would a.

be worse than one during the first division because all four meiotic products would be aneuploid. b. be better than the one during first division because only two of the four meiotic products would be aneuploid. c. have the same outcome as the first division, with all four products aneuploid. d. have the same outcome as the first division, as only two products would be aneuploid.

SYNTHESIZE 1. Color blindness is caused by a sex-linked, recessive gene. If a woman, whose father was color blind, marries a man with normal color vision, what percentage of their children will be color blind? What percentage of male children? Of female children? 2. Assume that the genes for seed color and seed shape are located on the same chromosome. A plant heterozygous for both genes is testcrossed to wrinkled green with the following results: green, wrinkled 645 green, round   36 yellow, wrinkled   29 yellow, round 590 What were the genotypes of the parents, and how far apart are these genes? 3. A low frequency of calico cats are male (about 1/3000). How could this arise?

when they are far enough apart for two crossovers to occur. when they are far enough apart that odd numbers of crossovers are about equal to even. only if recombination is low for that chromosome. only if the genes show genomic imprinting.

chapter

13 The Chromosomal Basis of Inheritance, and Human Genetics 267

14 CHAPTER

DNA: The Genetic Material Chapter Contents 14.1

The Nature of the Genetic Material

14.2

DNA Structure

14.3

Basic Characteristics of DNA Replication

14.4

Prokaryotic Replication

14.5

Eukaryotic Replication

14.6

DNA Repair

Molekuul/SPL/age fotostock

Introduction

Visual Outline Genetic material

if damaged

DNA repair

T T A

A

DNA structure

DNA replication

consists of

is

Nucleotides

Semiconservative

Template

is

Complementary strand

Semidiscontinuous

Building blocks

are

dNTPs

Enzymes

in include

Visible light

requires

T A

T A

join into Polarity

have

H N

H

O

N

G

Sugar

H

N

N

H

N

N

H

O

N

H

Polynucleotides

H

C N

N

with

which form

H

Double helix

Sugar

H

Leading strand

Lagging strand

H H

N N

Sugar

A

N

O

H

N

CH3 H

T N

N H

O

Sugar

Basepairing

joined by

Antiparallel strands

5′ 3′

DNA Pol

The rediscovery of Mendel at the turn of the 20th century led to the discovery of the genetic mechanisms detailed in chapters 12 and 13. One question not directly answered for more than 50 years was perhaps the simplest: What are genes made of? Genes were known to be on chromosomes, but they are complex structures composed of DNA, RNA, and protein. This chapter describes the chain of experiments that led to the model for DNA pictured here, and to the molecular mechanisms of heredity. This model was proposed by James Watson and Francis Crick, but was based on the data from many others. These experiments are among the most elegant in science and represent the dawn of a molecular era still with us today.

Griffith finds that bacterial cells can be transformed

14.1 The

Nature of the Genetic Material

The first clue came in 1928 with the work of the British microbiologist Frederick Griffith. Griffith was trying to make a vaccine that would protect against influenza, which was thought at the time to be caused by the bacteria Streptococcus pneumoniae. There are two forms of this bacteria: the normal virulent form that causes pneumonia, and a mutant, nonvirulent form that does not. The normal virulent form of this bacterium is referred to as the S form because it forms smooth colonies on a culture dish. The mutant, nonvirulent form, which lacks an enzyme needed to manufacture the polysaccharide coat, is called the R form because it forms rough colonies. Griffith performed a series of simple experiments in which mice were infected with these bacteria, then monitored for disease symptoms (figure 14.1). Mice infected with the virulent S form died from pneumonia, whereas infection with the nonvirulent R form had no effect. This result shows that the polysaccharide coat is necessary for virulence. If the virulent S form is first killed with heat, infection does not harm the mice, showing that the coat itself is not sufficient to cause disease. Lastly, infecting mice with a mixture of heat-killed S form with live R form caused pneumonia and death in the mice. This was unexpected, as neither treatment alone caused disease. Furthermore, high levels of live S form bacteria were found in the lungs of the dead mice. Somehow, the information specifying the polysaccharide coat had passed from the dead, virulent S bacteria to the live, coatless R bacteria in the mixture, permanently changing the coatless R bacteria into the virulent S variety. Griffith called this

Learning Outcomes 1. Describe the experiments of Griffith and Avery. 2. Evaluate the evidence for DNA as genetic material.

In chapters 12 and 13, you learned about the nature of inheritance and how genes, which contain the information to specify traits, are located on chromosomes. This finding led to the question of what component of chromosomes actually contains genetic information. Specifically, biologists wondered which molecule carries genetic information. They knew that chromosomes are composed primarily of both protein and DNA. Which of these organic molecules actually makes up the genes? Starting in the late 1920s and continuing for about 30  years, a series of investigations addressed this question. DNA consists of four chemically similar nucleotides. In contrast, protein contains 20 different amino acids that are much more chemically diverse than nucleotides. These characteristics seemed initially to indicate greater informational capacity in protein than in DNA. However, experiments began to reveal evidence in favor of DNA. We describe three of those major findings in this section.

Live Nonvirulent Strain of S. pneumoniae

Live Virulent Strain of S. pneumoniae

Mixture of Heat-Killed Virulent and Live Nonvirulent Strains of S. pneumoniae

Heat-Killed Virulent Strain of S. pneumoniae

+

Polysaccharide coat

Mice die

a.

Mice live

b.

Mice live

c.

Mice die Their lungs contain live pathogenic strain of S. pneumoniae

d.

Figure 14.1 Griffith’s experiment. Griffith was trying to make a vaccine against pneumonia and instead discovered transformation.

 a. Injecting live virulent bacteria into mice produces pneumonia. Injection of nonvirulent bacteria (b) or heat-killed virulent bacteria (c) had no effect. d. However, a mixture of heat-killed virulent and live nonvirulent bacteria produced pneumonia in the mice. This indicates the genetic information for virulence was transferred from dead, virulent cells to live, nonvirulent cells, transforming them from nonvirulent to virulent. chapter

14 DNA: The Genetic Material 269

2. When spun at high speeds in an ultracentrifuge, it migrated to the same level (density) as DNA. 3. Extracting lipids and proteins did not reduce transforming activity. 4. Protein-digesting enzymes did not affect transforming activity, nor did RNA-digesting enzymes. 5. DNA-digesting enzymes destroyed all transforming activity.

transfer of virulence from one cell to another ­transformation. Our modern interpretation is that genetic material was actually transferred between the cells.

Avery, MacLeod, and McCarty identify the transforming principle The agent responsible for transforming Streptococcus went undiscovered until 1944. In a classic series of experiments, O ­ swald Avery and his coworkers Colin MacLeod and Maclyn McCarty identified the substance responsible for transformation in Griffith’s experiment. They first prepared the mixture of dead S Streptococcus and live R Streptococcus that Griffith had used. Then they removed as much of the protein as they could from their preparation, eventually achieving 99.98% purity. They found that despite the removal of nearly all protein, the transforming activity was not reduced. Moreover, the properties of this substance resembled those of DNA in several ways: 1. The elemental composition agreed closely with that of DNA.

These experiments supported the identity of DNA as the substance transferred between cells by transformation and indicated that the genetic material, at least in this bacterial species, is DNA.

Hershey and Chase demonstrate that phage genetic material is DNA Avery’s results were not widely accepted at first because many biologists continued to believe that proteins were the genetic material. But additional evidence supporting Avery’s conclusion was provided in 1952 by Alfred Hershey and Martha

SCIENTIFIC THINKING Hypothesis: DNA is the genetic material in bacteriophage. Prediction: The phage life cycle requires reprogramming the cell to make phage proteins. The information for this must be introduced into the cell during infection. Test: Because DNA, but not protein, contains phosphate, and protein, but not DNA, contains sulfur, we can label each specifically with either radioactive phosphate (32P for DNA), or radioactive sulfur (35S for protein). This allows us to follow each molecule separately. Phage are grown on either 35S or 32P, then used to infect cells in two experiments. The phage heads are removed by brief agitation in a blender, and centrifuged to separate cells (pellet) from phage heads (supernatant) and each can be assayed for the radioactive labels. 35S-Labeled

Bacteriophages

+

Phage grown in radioactive 35S, which is incorporated into phage coat

Viruses infect bacteria 32P-Labeled

Blender separates phage coat from bacteria

Centrifuge forms bacterial pellet

35S

in supernatant

Bacteriophages

+

Phage grown in radioactive 32P, which is incorporated into phage DNA

Viruses infect bacteria

Blender separates phage coat from bacteria

Centrifuge forms bacterial pellet

32P

in bacterial pellet

Result: When the experiment is done, only 32P makes it into the cell in any significant quantity. Conclusion: Thus, DNA must be the molecule that is used to reprogram the cell. Further Experiments: How does this experiment complement or extend the work of Avery on the identity of the transforming principle?

Figure 14.2 Hershey–Chase experiment showed DNA is genetic material for phage. 270 part III Genetic and Molecular Biology

Chase, who experimented with viruses that infect bacteria. These viruses are called bacteriophages, or more simply, phages. Viruses, described in more detail in chapter 26, are much simpler than cells; they generally consist of genetic material (DNA or RNA) surrounded by a protein coat. When a phage infects a bacterial cell, it first binds to the cell’s outer surface and then injects its genetic information into the cell. Once inside the cell, the viral genetic information uses the cell’s informationprocessing machinery to make viral proteins, leading to the production of thousands of new viruses. The buildup of viruses eventually causes the cell to burst, or lyse, releasing progeny phage. The phage used by Hershey and Chase contains only DNA and protein, providing the simplest possible system to differentiate the roles of DNA and protein. If they could identify the molecule injected into the cell, they would identify the viruses’ genetic material. To do this, they needed a method to introduce unique labels into both DNA and protein. Nucleotides contain phosphorus, but proteins do not, and some amino acids ­contain sulfur, but DNA does not. Thus, the radioactive 32P isotope will label DNA specifically, and the isotope 35S will label proteins specifically. The two isotopes are easily distinguished based on the particles they emit when they decay. Two experiments were performed (figure 14.2). In one, viruses were grown on a medium containing 32P, which was incorporated into DNA; in the other, viruses were grown on medium containing 35S, which was incorporated into coat proteins. Each culture of labeled viruses was then used to infect separate bacterial cultures. After infection, the bacterial cell suspension was agitated in a blender to remove the infecting viral particles from the surfaces of the bacteria. This step ensured that only the part of the virus that had been injected into the bacterial cells—that is, the genetic material—would be detected. Each bacterial suspension was then centrifuged to produce a pellet of cells for analysis. In the 32P experiment, a large amount of radioactive phosphorus was found in the cell pellet, but in the 35 S experiment, very little radioactive sulfur was found in the pellet (figure 14.2). Hershey and Chase deduced that the genetic information viruses injected into bacteria was DNA, and not protein.

Learning Outcomes Review 14.1

Experiments with pneumonia-causing bacteria showed that virulence could be passed from one cell to another, a phenomenon termed transformation. When the factor responsible for transformation was purified, it was shown to be DNA. Labeling experiments with phage also indicated that the genetic material was DNA and not protein. ■■

Why was protein an attractive candidate for the genetic material?

14.2

DNA Structure

Learning Outcomes 1. Explain how the Watson–Crick structure rationalized the data available to them. 2. Evaluate the significance of complementarity for DNA structure and function.

DNA was discovered in 1869 by Friedrich Miescher. This was only four years after Mendel’s work was published, and 30 years before Mendel was rediscovered. Miescher extracted a white substance from the nuclei of white blood cells. He also isolated the same substance from salmon sperm. Analysis of this material, which was rich in phosphorus, indicated that it appeared to differ from other known cellular constituents. Miescher named this new biological substance “nuclein” because it was associated with the nucleus. Later, because nuclein was slightly acidic, it came to be called nucleic acid.

DNA’s components were known, but its three-dimensional structure was a mystery Although the three-dimensional structure of the DNA molecule was a mystery, the components of nucleic acids were known (figure 14.3): 1. A 5-carbon sugar 2. A phosphate (PO4 –) group 3. A nitrogen-containing (nitrogenous) base. This may be a two-ringed purine (adenine, A, or guanine, G), or a single-ringed pyrimidine (thymine, T, or cytosine, C); in RNA the pyrimidine uracil, U, replaces thymine, T). We need a way to be able to unambiguously refer to each carbon atom in a nucleotide. We can do this by adding a prime symbol ( ′ ) to carbon numbers in the sugar to distinguish these from atoms in the bases that are just numbered. The ribose sugars found in nucleic acids consist of a five-membered ring with four carbon atoms and an oxygen atom. As illustrated in figure 14.3, the carbon atoms are numbered 1′ to 5′, clockwise from the oxygen atom. The phosphate group is attached to the 5′ carbon atom of the sugar, and bases are attached to the 1′ carbon. In addition, a free hydroxyl (–OH) group is attached to the 3′ carbon atom. This means each nucleotide has a 5′ phosphate end, and a 3′ hydroxyl end. Nucleotide monomers are joined together by a dehydration reaction involving the 5′ phosphate of one nucleotide with the 3′ hydroxyl of another nucleotide. This linkage is called a phosphodiester bond because the phosphate group is now linked to the two sugars by means of a pair of ester bonds (figure 14.4). Literally hundreds of millions of nucleotides are joined together via these linkages to form the nucleic acid polymers in your DNA. Polynucleotide strands of DNA or RNA can be thought of as starting with a 5′ phosphate group and ending with a 3′ hydroxyl group. Therefore, every DNA and RNA molecule has an intrinsic polarity, and we can refer unambiguously to each end of the molecule. By convention, the sequence of bases is usually written in the 5′-to-3′ direction. chapter

14 DNA: The Genetic Material 271

Nitrogenous Base Nitrogenous base

1

9

2

N

H

C

N C N C H

3

CH2

C

N

N

C H

H

C

N C C

N

H

N C

C

NH2

H

Adenine

N

Guanine

5′

O−

1′

4′ 3′

2′

OH in RNA

OH

H in DNA

Sugar

H

C

H

C

C N

N C

O

O

NH2

O Pyrimidines

O

4

N

Purines

O P

N

8

Phosphate group

−O

6

5

O

NH2

NH2

7N

O

H Cytosine (both DNA and RNA)

H3C

C

H

C

C N

N

H

H

C

C

O

H

C

H Thymine (DNA only)

C N

N

H

C

O

H Uracil (RNA only)

Figure 14.3 Nucleotide subunits of DNA and RNA. The nucleotide subunits of DNA and RNA are composed of three components: a 5-carbon sugar (deoxyribose in DNA and ribose in RNA); a phosphate group; and a nitrogenous base (either a purine or a pyrimidine). PO4 Base

5′ CH2 O 1′

5′ 3′

C

1. The proportion of A always equals that of T, and the proportion of G always equals that of C, or: A = T, and G = C. 2. The ratio of G–C to A–T varies with different species.

2′

O Phosphodiester bond

−O

O

P O

Base

5′ CH2 O 1′

4′ 3′ OH

2′

Figure 14.4 A dinucleotide. The phosphodiester bond joining two nucleotides is highlighted, as are the 5’ and 3’ ends of the molecule.

Chargaff, Franklin, and Wilkins obtained some structural evidence To understand the model that Watson and Crick proposed, we need to review the evidence that they had available to construct their model.

Chargaff’s rules A careful study carried out by Erwin Chargaff showed that the nucleotide composition of DNA molecules varied in complex ways, depending on the source of the DNA. This strongly sug­gested that DNA was not a simple repeating polymer and that it might have the information-encoding properties genetic material requires. Despite DNA’s complexity, however, Chargaff observed an important 272 part III Genetic and Molecular Biology

underlying regularity in the ratios of the bases found in native DNA: the amount of adenine present in DNA always equals the amount of thymine, and the amount of guanine always equals the amount of cytosine. Two important findings from this work are often called Chargaff’s rules:

As mounting evidence indicated that DNA stored the hereditary information, investigators began to puzzle over how such a seemingly simple molecule could carry out such a complex coding function.

X-ray diffraction patterns of DNA The technique of X-ray diffraction provided more direct information about the possible structure of DNA. In X-ray diffraction, crystals of a molecule are bombarded with a beam of X-rays. The rays are bent, or diffracted, by the molecules they encounter, and the diffraction pattern is recorded on photographic film. The patterns resemble the ripples created by tossing a rock into a smooth lake. When analyzed mathematically, the diffraction pattern can yield information about the three-dimensional structure of a molecule. The problem with using this technique with DNA was that in the 1950s, it was impossible to obtain true crystals of natural DNA. However, British researcher Maurice Wilkins learned how to prepare uniformly oriented DNA fibers, and with graduate student Ray Gosling succeeded in obtaining the first crude diffraction information on natural DNA in 1950. Their early X-ray photos suggested that the DNA molecule has the shape of a helix. The British chemist Rosalind Franklin (figure 14.5a) continued this work, perfecting the technique to obtain ever clearer “pictures” of the oriented DNA fibers. The clearest of these images (figure 14.5b) confirmed that DNA was a helix, and allowed calculation of the dimensions of the molecule, indicating a diameter of about 2 nm and a complete helical turn every 3.4 nm.

a.

b.

Figure 14.5 Rosalind Franklin’s X-ray diffraction patterns. a. Rosalind Franklin. b. This X-ray diffraction photograph of DNA fibers, made in 1953 by Rosalind Franklin, was interpreted to show the helical structure of DNA.  (a) Jewish Chronicle Archive/Heritage Image Partnership Ltd/Alamy Stock Photo; (b) Omikron/Science Source

Tautomeric forms of bases One piece of evidence important to Watson and Crick was the form of the bases themselves. Because of the alternating double and single bonds in the bases, they actually exist in equilibrium between two different forms when in solution. The different forms have to do with keto (C=O) versus enol (C–OH) groups and amino (–NH2) versus imino (=NH) groups that are attached to the bases. These structural forms are called tautomers. The importance of this distinction is that the two forms have very different hydrogen-bonding possibilities. The predominant forms of the bases contain the keto and amino groups (see figure 14.3), but a prominent biochemistry text of the time actually contained the opposite, and incorrect, information. Legend has it that Watson learned the correct forms while having lunch with a biochemist friend.

The Watson–Crick model fits the available evidence Learning informally of Franklin’s results before they were published in 1953, American chemist James Watson and English molecular biologist Francis Crick, two young investigators at Cambridge University, quickly worked out a likely structure for the DNA molecule (figure 14.6), which we now know was substantially correct. Watson and Crick did not perform a single experiment themselves related to DNA structure; rather, they built detailed molecular models based on the information available. The key to the model was their understanding that each DNA molecule is actually made up of two chains of nucleotides that are intertwined—the double helix.

Figure 14.6 The DNA double helix. James Watson (left)

and Francis Crick (right) deduced the structure of DNA in 1953 from Chargaff’s rules, knowing the proper tautomeric forms of the bases and using Franklin’s diffraction studies. Barrington Brown/Science Source

5′

Phosphate group

P

5′

O 1′

4′ 3′

Phosphodiester bond

2′

P

5′

O

4′

1′ 3′

P

2′

5′

O

1′

4′ 3′

5-carbon sugar

2′

Nitrogenous base P

5′

O 3′

The phosphodiester backbone The two strands of the double helix are made up of long polymers of nucleotides, and as described earlier in this section, each strand is made up of repeating sugar and phosphate units joined by phosphodiester bonds (figure 14.7). We call this the phosphodiester

1′

4′

OH

2′

3′

Figure 14.7 Structure of a single strand of DNA. The

phosphodiester backbone is composed of alternating sugar and phosphate groups. The bases are attached to each sugar. chapter

14 DNA: The Genetic Material 273

2 nm

5′

backbone of the molecule. The two strands of the backbone are then wrapped about a common axis forming a double helix (figure 14.8). The helix is often compared to a spiral staircase, in which the two strands of the double helix are the handrails on the staircase.

3′

Complementarity of bases

A

T G Minor groove

3.4 nm

C

T G T

A

0.34 nm

C

G Major groove

G A G

T

Watson and Crick proposed that the two strands were held together by hydrogen bonds between bases on opposite strands. The nature of these hydrogen bonds results in specific base-pairs: adenine (A) can form two hydrogen bonds with thymine (T) in an A–T base-pair, and guanine (G) can form three hydrogen bonds with cytosine (C) in a G–C base-pair (figure 14.9). Note that each base-pair combines a two-ringed purine with a single-ringed pyrimidine, which means that the diameter of each base-pair is the same. A purine–purine base-pair would be wider, and a pyrimidine–pyrimidine base-pair would be narrower. In either case, this would distort the double helix formed by base-pairs. We call this pattern of base-pairing complementary, and this is a critical feature of all aspects of DNA function. The two strands of the DNA molecule are not identical, but each strand specifies the other because of complementarity of base-pairs. This means that if you know one strand of a DNA double helix, you know the other strand, even though they are not the same. For instance, the DNA strand: 5’ AGGCTATGCTAAGCTCCTTAA 3’ will have the complementary sequence: 3’ TCCGATACGATTCGAGGAATT 5’

C Hydrogen bond N

H N

G

Sugar

H

O

H N

N

H

N

N

H

O

H H

C N

N

Major groove

Sugar

H Hydrogen bond

H H Minor groove

N

N

N Sugar

A

N

H

O

H

N

CH3 H

T N

N H

O

Sugar

Figure 14.9 Base-pairing holds strands together.  3′

5′

Figure 14.8 The double helix. Shown with the phosphodiester backbone as a ribbon on top and a space-filling model on the bottom. The bases protrude into the interior of the helix where they hold it together by base-pairing. The backbone forms two grooves, the larger major groove and the smaller minor groove. 274 part III Genetic and Molecular Biology

The hydrogen bonds that form between A and T and between G and C are shown with dashed lines. These produce A–T and G–C basepairs that hold the two strands together. This always pairs a purine with a pyrimidine, keeping the diameter of the double helix constant.

Data analysis Explain how the Watson–Crick model accounts for the data discussed in the text.

We will see this principle of complementarity in action as we learn about DNA replication and expression in section 14.3. The Watson–Crick model also explains Chargaff’s results: the reason that the amount of A equals T, and the amount of G equals C, is that A base-pairs with T, and G base-pairs with C. The relative amounts of A/T and G/C can vary, but the amount of A must be equal to T, and the amount of G must be equal to C because of complementary base-pairing.

Antiparallel configuration As stated earlier in this section, a single phosphodiester strand has an inherent polarity, meaning that one end terminates in a 3′ OH and the other end terminates in a 5′ PO4. Strands are thus referred to as having either a 5′-to-3′ or a 3′-to-5′ polarity. Two strands could be put together in two ways: with the polarity the same in each (parallel) or with the polarity opposite (antiparallel). Native double-stranded DNA always has the antiparallel configuration, with one strand running 5′ to 3′ and the other running 3′ to 5′ (see figure 14.8). In addition to its complementarity, this antiparallel nature also has important implications for DNA replication.

The Watson–Crick DNA molecule In the Watson and Crick model, a DNA molecule consists of two phosphodiester strands wrapped about a common helical axis, with the bases extending to the interior of the helix. The sequence of bases on the two strands is complementary, so they form base-pairs that hold the strands together. Each strand is also polar, with a 5′ and a 3′ end, and they are oriented in the opposite (antiparallel) direction (see figures 14.8 and 14.9). Although each individual hydrogen bond is low energy, the sum of thousands, or millions, of such bonds makes a very stable molecule. This also means that a DNA molecule can have regions where the helix can be “opened” up without affecting the stability of the entire molecule, which is critical for its function. Although the Watson–Crick model provided a rational structure for DNA, researchers had to answer further questions about how DNA could be replicated, a crucial step in cell division, and also about how cells could repair damaged or otherwise altered DNA. We explore these questions in the rest of this chapter. (In chapter 15, we continue with the genetic code and the connection between the code and protein synthesis.)

Learning Outcomes Review 14.2

Chargaff showed that in DNA, the amount of adenine was equal to the amount of thymine, and the amount of guanine was equal to that of cytosine. X-ray diffraction studies by Franklin and Wilkins indicated that DNA formed a helix. Watson and Crick built a model consisting of two antiparallel strands wrapped in a helix about a common axis. The two strands are held together by hydrogen bonds between the bases: adenine pairs with thymine and guanine pairs with cytosine. The two strands are thus complementary to each other. ■■

Why was information about the proper tautomeric form of the bases critical?

14.3

Basic Characteristics of DNA Replication

Learning Outcomes 1. Illustrate the products of semiconservative replication. 2. Describe the requirements for DNA replication.

The accurate replication of DNA prior to cell division is a basic and crucial function. Research has revealed that this complex process requires the participation of a large number of cellular p­ roteins. ­Before geneticists could look for these details, however, they needed­to perform some groundwork on the general mechanisms.

Meselson and Stahl demonstrated the semiconservative mechanism The Watson–Crick model immediately suggested that the basis for copying the genetic information is complementarity. One chain of the DNA molecule may have any conceivable base sequence, but this sequence completely determines the sequence of its partner in the duplex. In replication, the sequence of parental strands must be duplicated in daughter strands. That is, one parental helix with two strands must yield two daughter helices with four strands. The two daughter molecules are then separated during the course of cell division. Three models of DNA replication are possible (figure 14.10): 1. In a conservative model, both strands of the parental duplex would remain intact (conserved), and new DNA copies would consist of all-new molecules. Both daughter strands would contain all-new molecules. 2. In a semiconservative model, one strand of the parental duplex would remain intact in each daughter duplex (semiconserved); a new complementary strand would be synthesized with each parental strand. Each daughter duplex would consist of one parental strand and one newly synthesized strand. 3. In a dispersive model, copies of DNA would consist of mixtures of parental and newly synthesized strands; that is, the new DNA would be dispersed throughout each strand of both daughter molecules after replication. Notice that these three models suggest general mechanisms of replication, without specifying any molecular details of the process.

The Meselson–Stahl experiment The three models for DNA replication were evaluated in 1958 by Matthew Meselson and Franklin Stahl. To distinguish between these models, they labeled DNA and then followed the labeled DNA through two rounds of replication (figure 14.11). The label Meselson and Stahl used was a heavy isotope of nitrogen (15N), not a radioactive label. Molecules containing 15N chapter

14 DNA: The Genetic Material 275

DNA E. coli 15N medium

E. coli cells grown in 15N medium

14N medium

Cells shifted to 14N medium and allowed to grow

Samples taken at three time points and suspended in cesium chloride solution 0 min 0 rounds

20 min 1 round

40 min 2 rounds

Samples are centrifuged

Conservative

Semiconservative

Dispersive

Figure 14.10 Three possible models for DNA replication. The conservative model produces one entirely new

molecule and conserves the old. The semiconservative model produces two hybrid molecules of old and new strands. The dispersive model produces hybrid molecules with each strand a mixture of old and new.

0 rounds

1 round

2 rounds

0

have a greater density than those containing the common 14N isotope. Ultracentrifugation can be used to separate molecules that have different densities. Bacteria were grown in a medium containing 15N, which became incorporated into the bases of the bacterial DNA. After several generations, the DNA of these bacteria was denser than that of bacteria grown in a medium containing the normally available 14N. Meselson and Stahl then transferred the bacteria from the 15N medium to 14N medium and collected the DNA at various time intervals. The DNA for each interval was dissolved in a solution containing a heavy salt, cesium chloride. This solution was spun at very high speeds in an ultracentrifuge. The enormous centrifugal forces caused cesium ions to migrate toward the bottom of the centrifuge tube, creating a gradient of cesium concentration, and thus of density. Each DNA strand floated or sank in the gradient until it reached the point at which its density exactly matched the density of the cesium at that location. Because 15N strands are denser than 14 N strands, they migrated farther down the tube. The DNA collected immediately after the transfer of bacteria to new 14N medium was all of one density equal to that of 15N DNA alone. However, after the bacteria completed a first round of DNA replication, the density of their DNA had decreased to a value 276 part III Genetic and Molecular Biology

1

2 Top

Bottom

Rounds of replication

Figure 14.11 The Meselson–Stahl experiment. Bacteria grown

in heavy 15N medium are shifted to light 14N medium and grown for two rounds of replication. Samples are taken at time points corresponding to zero, one, and two rounds of replication and centrifuged in cesium chloride to form a gradient. The actual data are shown at the bottom with the interpretation of semiconservative replication shown schematically.  Meselson, M., Stahl, F., (1958) “The replication of DNA in Escherichia coli,” PNAS, 44(7):671-682, Fig. 4a

Data analysis What would you predict for the products of a third round of replication?

intermediate between 14N DNA alone and 15N DNA. After the second round of replication, two density classes of DNA were observed: one intermediate and one equal to that of 14N DNA (figure 14.11).

1 1 2̸ light (new) and 2̸ heavy (old) molecules. But after two rounds of replication, the dispersive model would still yield only a single density; DNA strands would be composed of 3 4̸ light and 1 4̸ heavy molecules. Instead, two densities were observed. Therefore, this model is also rejected.

Interpretation of the Meselson–Stahl findings Meselson and Stahl compared their experimental data with the results that would be predicted on the basis of the three models: 1. The conservative model was not consistent with the data because after one round of replication, two densities should have been observed: DNA strands would either be all-heavy (parental) or all-light (daughter). This model is rejected. 2. The semiconservative model is consistent with all observations: After one round of replication, a single density would be predicted because all DNA molecules would have a light strand and a heavy strand. After two rounds of replication, half of the molecules would have two light strands, and half would have a light strand and a heavy strand—and so two densities would be observed. Therefore, the results support the semiconservative model. 3. The dispersive model was consistent with the data from the first round of replication, because in this model, every DNA helix would consist of strands that are mixtures of Template Strand

Replication requires three things: something to copy, something to do the copying, and the building blocks to make the copy. The parental DNA molecules serve as a template, enzymes perform the actions of copying the template, and the building blocks are nucleotides. Note that nucleotides have one, two, or three phosphate groups, which are called nucleoside mono, di and triphosphates. The nucleoside triphosphates are the actual building blocks, and during synthesis, the two terminal phosphates are cleaved, providing energy for the reaction (figure 14.12). Template Strand

5′ C

O

O

P

T

A

P

T

O

A

O

O P

P

A

P

A

DNA polymerase III

O

T

O

O

T

O P

P C O

P

C

O

G

O

P O

G

P

P 3′

P

OH

A O

A P

T

P

5′

P

G

P

P

P

P

5′ C

O

O

O

New Strand

3′

HO P

G

O

Sugar– phosphate backbone

DNA replication requires a template, nucleotides, and a polymerase enzyme

New Strand

3′

HO

The basic mechanism of DNA replication is semiconservative. At the simplest level, then, DNA is replicated by opening up a DNA helix and making copies of both strands to produce two daughter helices, each consisting of one old strand and one new strand.

P

O

P

O

T

P

Pyrophosphate

P

O

3′

A OH

P

O P

OH

A

5′

Figure 14.12 Action of DNA polymerase. DNA polymerases add nucleotides to the 3′ end of a growing chain. The nucleotide added

depends on the base that is in the template strand. Each new base must be complementary to the base in the template strand. With the addition of each new nucleoside triphosphate, two of its phosphates are cleaved off as pyrophosphate.

?

Inquiry question Why do you think it is important that the sugar–phosphate backbone of DNA is held together by covalent bonds, and the cross-bridges between the two strands are held together by hydrogen bonds?

chapter

14 DNA: The Genetic Material 277

DNA replication has a beginning, where the process starts; a middle, where building blocks are added; and an end, where the process is finished. For biochemical processes like replication, we call this initiation, elongation, and termination. Although these divisions may seem arbitrary, in fact, discrete functions are usually required for initiation and termination that are not necessary for elongation. A number of enzymes work together to accomplish the task of assembling a new strand, but the enzyme that actually matches the existing DNA bases with complementary nucleotides and then links the nucleotides together to make the new strand is DNA polymerase (figure 14.12). All DNA ­polymerases that have been examined have several common features. They all add new bases to the 3′ end of existing strands. That is, they synthesize in a 5′-to-3′ direction by extending a strand base-paired to the template. All DNA polymerases also require a primer to begin synthesis; they cannot begin without a strand of RNA or DNA base-paired to the template. RNA polymerases do not have this requirement, so they usually synthesize the primers. 5′ 3′

5′

RNA polymerase makes primer

5′

3′

3′

5′

DNA polymerase extends primer

Learning Outcomes Review 14.3

Meselson and Stahl showed that the basic mechanism of replication is semiconservative: each new DNA helix is composed of one old strand and one new strand. The process of replication requires a template to copy, nucleoside triphosphate building blocks, and the enzyme DNA polymerase. DNA polymerases synthesize DNA in a 5′-to-3′ direction from a primer, usually RNA. ■■

In the Meselson–Stahl experiment, what would the results be if the DNA were denatured prior to separation by ultracentrifugation?

14.4

Prokaryotic Replication

Learning Outcomes 1. Describe the functions of E. coli DNA polymerases. 2. Explain why replication is discontinuous on one strand. 3. Diagram the functions found at the replication fork.

To build up a more detailed picture of replication, we first concentrate on prokaryotic replication using E. coli as a model system. We can then look at eukaryotic replication primarily in terms of deviating from prokaryotic replication. There are a number of reasons why E. coli has been such an effective model system to study replication: it has a small genome (4.6 million bp of DNA); it has a large collection of mutants that affect replication; and the single circular chromosome is replicated at up to 1,000 nucleotides per second.

Prokaryotic replication starts at a single origin In E. coli, replication begins at a specific origin (called oriC) when a protein called DnaA recognizes and binds oriC. This causes an AT-rich sequence to be melted, making single strands available to load the enzymatic machinery for replication. An AT-rich region is easier to melt, as A–T base-pairs have only two hydrogen bonds compared to the three in G–C base-pairs. After initiation, replication proceeds bidirectionally from this unique origin to the unique terminus (figure 14.13). We call the DNA controlled by an origin a replicon. In this case, the chromosome plus the origin forms a single replicon.

 . coli has at least three different E DNA polymerases As mentioned in section 14.3, DNA polymerase refers to a class of enzymes that use a DNA template to assemble a new complementary strand. The first DNA polymerase isolated in E. coli was DNA polymerase I (Pol I). At first, investigators assumed this polymerase was all that was required for DNA replication. Later, a mutant was isolated with no Pol I activity, but that could still replicate its chromosome. Two additional polymerases were isolated from this strain of E. coli: DNA polymerase II (Pol II) and DNA polymerase III (Pol III). As with all known DNA polymerases, all three of these enzymes synthesize polynucleotide strands only in the 5′-to-3′ direction and require a primer. In addition to adding nucleotides to a growing DNA strand, some polymerases can also remove nucleotides, or act as a nuclease. Enzymes that act as nucleases are classified as either endonucleases (which cut DNA internally) or exonucleases (which remove nucleo­tides from the end of DNA). DNA Pol I, Pol II, and Pol III have 3′-to-5′ exonuclease activity, which serves as a proofreading function because it allows the enzyme to “back up” and remove a mispaired base. DNA Pol I also has a 5′-to-3′ exonuclease activity, which can be used to remove RNA primers. The three different polymerases have different roles in the replication process. DNA Pol III is the main replication enzyme; it is responsible for the bulk of DNA synthesis. DNA Pol I acts on Origin

Replisome Origin

Termination

Termination

Termination Origin

Termination Replisome

Termination Origin

Origin

Figure 14.13 Replication is bidirectional from a unique origin. Replication initiates from a unique origin. Two separate replisomes are loaded onto the origin and initiate synthesis in the opposite directions on the chromosome. These two replisomes continue in opposite directions until they come to a unique termination site. 278 part III Genetic and Molecular Biology

Unwinding DNA requires energy and causes torsional strain

Supercoiling Replisomes

No Supercoiling Replisomes DNA gyrase

Figure 14.14 Unwinding the helix causes torsional strain. 

Although most DNA polymerases can unwind DNA during synthesis, replication is more efficient if the helix is unwound ahead of the polymerase. An enzyme with DNA unwinding activity is called a helicase. This process requires energy in the form of ATP, and can progressively unwind DNA, forming single strands. These single strands are not stable because the hydrophobic bases are exposed to water. Cells solve this problem with another protein, single-strandbinding protein (SSB), that will coat exposed single strands. The unwinding of the two strands introduces torsional strain in the DNA molecule. Imagine two rubber bands twisted together. If you now unwind the rubber bands, what happens? The rubber bands, already twisted about each other, will further coil in space. When this happens with a DNA molecule it is called supercoiling (figure 14.14). The branch of mathematics that studies how forms twist and coil in space is called topology, and therefore we describe this coiling of the double helix as the topological state of DNA. This state describes how the double helix itself coils in space. You have already seen an example of this coiling with DNA wrapped about histone proteins in the nucleosomes of eukaryotic chromosomes (see chapter 10). Enzymes that can alter the topological state of DNA are called topoisomerases. Topoisomerase enzymes act to relieve the torsional strain caused by unwinding and to prevent this supercoiling from happening. DNA gyrase is the topoisomerase involved in DNA replication (figure 14.14).

If the ends of a linear DNA molecule are constrained, as they are in the cell, unwinding the helix produces torsional strain. This can cause the double helix to further coil in space (supercoiling). The enzyme DNA gyrase can relieve supercoiling.

Replication is semidiscontinuous

the lagging strand to remove primers and replace them with DNA. The Pol II enzyme does not appear to play a role in replication but is involved in DNA repair processes. For many years, these three polymerases were thought to be the only DNA polymerases in E. coli, but several others have been identified. There are now five known polymerases, although not all are active in DNA replication.

Earlier, DNA was described as being antiparallel—meaning that one strand runs in the 3′-to-5′ direction, and its complementary strand runs in the 5′-to-3′ direction. The antiparallel nature of DNA combined with the nature of the polymerase enzymes puts constraints on the replication process. Because polymerases can synthesize DNA in only one direction, and the two DNA strands run in opposite directions, polymerases on the two strands must be synthesizing DNA in opposite directions (figure 14.15).

5′

Figure 14.15 Replication is semidiscontinuous. The 5'-to-3'

3′

5′

5′

Open helix and replicate

3′

Lagging strand (discontinuous)

First RNA primer Open helix and replicate further 3′

3′

Second RNA primer

5′ RNA primer

5′

3′

3′

synthesis of the polymerase and the antiparallel nature of DNA mean that only one strand, the leading strand, can be synthesized continuously. The other strand, the lagging strand, must be made in pieces, each with its own primer.

5′

Leading strand (continuous) RNA primer 5′ 3′ chapter

14 DNA: The Genetic Material 279

The requirement of DNA polymerases for a primer means that on one strand primers need to be added as the helix is opened up (figure 14.15). This means that one strand can be synthesized in a continuous fashion from an initial primer, but the other strand must be synthesized in a discontinuous fashion with multiple priming events and short sections of DNA being assembled. The strand that is continuous is called the leading strand, and the strand that is discontinuous is the lagging strand. DNA fragments synthesized on the lagging strand are named Okazaki fragments in honor of the man who first experimentally demonstrated discontinuous synthesis. They introduce a need for even more enzymatic activity on the lagging strand, as is described next.

Synthesis occurs at the replication fork The partial opening of a DNA helix to form two single strands has a forked appearance, and is thus called the replication fork. All of the enzymatic activities that we have discussed plus a few more are found at the replication fork (table 14.1). Synthesis on the leading strand and on the lagging strand proceed in different ways, however.

Priming The primers required by DNA polymerases during replication are synthesized by the enzyme DNA primase. This enzyme is an RNA polymerase that synthesizes short stretches of RNA 10 to 20 bp (base-pairs) long that function as primers for DNA polymerase. Later on, the RNA primer is removed and replaced with DNA.

indefinitely by the action of DNA Pol III. If the enzyme remains attached to the template, it can synthesize around the entire circular E. coli chromosome. The ability of a polymerase to remain attached to the template is called processivity. The Pol III enzyme is a large multisubunit enzyme that has high processivity due to the action of one subunit of the enzyme, called the β subunit (figure 14.16a). The β subunit is made up of two identical protein chains that come together to form a circle. This circle can be loaded onto the template like a clamp to hold the Pol III enzyme to the DNA (figure 14.16b). This structure is therefore referred to as the “sliding clamp,” and a similar structure is found in eukaryotic polymerases as well. For the clamp to function, it must be opened and then closed around the DNA. A multisubunit protein called the clamp loader accomplishes this task. This function is also found in eukaryotes.

Lagging-strand synthesis Because synthesis on the lagging strand is discontinuous, more steps are required to replicate this strand. Primase is needed to synthesize primers for each Okazaki fragment, and then all these RNA primers must be removed and replaced with DNA. Finally, the fragments need to be stitched together. The Okazaki fragments themselves are synthesized by DNA Pol III, like the leading strand, but the primers are removed and replaced with DNA by DNA Pol I. With its 5′-to-3′ exonuclease activity, Pol I can remove primers and then replace them with its

Leading-strand synthesis Synthesis on the leading strand is relatively simple. A single priming event is required, and then the strand can be extended TA B L E 1 4 .1 Protein Helicase

DNA Replication Enzymes of E. coli Role Unwinds the double helix

Size (kDa)

Molecules per Cell

300

20

a.

Primase

Synthesizes RNA primers

60

50

Single-strand-binding protein

Stabilizes singlestranded regions

74

300

DNA gyrase

Relieves torque

400

250

DNA polymerase III

Synthesizes DNA

≈900

20

DNA polymerase I

Erases primer and fills gaps

103

300

DNA ligase

Joins the ends of DNA segments; DNA repair

74

300

280 part III Genetic and Molecular Biology

b.

Figure 14.16 The DNA polymerase sliding clamp. a. The β subunit forms a ring that can encircle DNA. b. The β subunit is shown attached to the DNA. This forms the “sliding clamp” that keeps the polymerase attached to the template.  Ramon Andrade 3Dciencia/SPL/Science Source

5′

?

DNA ligase Lagging strand (discontinuous) RNA primer

Termination occurs at a specific site located roughly opposite oriC on the circular chromosome. The last stages of replication produce two daughter molecules that are intertwined like two rings in a chain. These intertwined molecules are unlinked by the same enzyme that relieves torsional strain at the replication fork: DNA gyrase.

Okazaki fragment made by DNA polymerase III

Primase Leading strand (continuous)

The replisome contains all the necessary enzymes for replication

3′

Figure 14.17 Lagging-strand synthesis. The action of

primase synthesizes the primers needed by DNA polymerase III (not shown). These primers are removed by DNA polymerase I using its 5′-to-3′ exonuclease activity, then extending the previous Okazaki fragment to replace the RNA. The nick between Okazaki fragments after primer removal is sealed by DNA ligase.

usual polymerase activity. The Pol I extends the Okazaki fragment “behind” it, while removing the RNA primer “in front of ” it. This leaves only the last phosphodiester bond to be formed where synthesis by Pol I ends. This is done by DNA ligase, which seals this “nick,” acting to join the Okazaki fragments into complete strands. All of this activity on the lagging strand is summarized in figure 14.17.

Single-strand-binding proteins (SSB)

would happen to DNA replication in a cell where this enzyme is not functional?

Termination

DNA polymerase I

New bases

Inquiry question What is the role of DNA ligase? What

Cells have many macromolecular machines that perform specific functions, such as the ribosome that synthesizes proteins. The macromolecular machine responsible for DNA replication is the replisome. The replisome has two main subcomponents: the primosome, and a complex of two DNA Pol III enzymes, one for each strand. The primosome consists of primase and helicase, along with a number of accessory proteins. Despite our calling one strand the lagging strand, the two Pol III enzymes in the replisome are active on both strands simultaneously. How can the two strands be synthesized in the same direction when the strands are antiparallel? The best model involves a loop formed in the lagging strand, allowing the two polymerases to move in the same direction (figure 14.18). The two Pol III complexes include two synthetic core subunits, each with its own β sliding clamp subunit. The entire

β clamp (sliding clamp)

Figure 14.18 The replication fork. A model for

Leading strand 3′ 5′

Clamp loader

DNA gyrase Open β clamp

5′ 3′ Parent DNA

Helicase Primase

DNA polymerase III

Lagging strand Okazaki fragment

New bases 3′ 5′

DNA polymerase I DNA ligase RNA primer

the structure of the replication fork with two polymerase III enzymes held together by a large complex of accessory proteins. These include the “clamp loader,” which loads the β subunit sliding clamp periodically on the lagging strand. The polymerase III on the lagging strand periodically releases its template and reassociates along with the β clamp. The loop in the laggingstrand template allows both polymerases to move in the same direction despite DNA being antiparallel. Primase, which makes primers for the laggingstrand fragments, and helicase are also associated with the central complex. Polymerase I removes primers and ligase joins the fragments together.

chapter

14 DNA: The Genetic Material 281

Leading strand

DNA polymerase III Helicase

Clamp loader

DNA gyrase 5′ 3′

3′ 5′

3′ 5′ Clamp loader

5′ 3′

DNA ligase patches “nick”

RNA primer

RNA primer β clamp

Primase Single-strand binding-proteins (SSB)

Lagging strand RNA primer

4. The clamp loader attaches the β clamp and transfers this to

polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers.

1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand.

3′ 5′

3′ 5′

Leading strand replicates continuously

5′ 3′

5′ 3′ First Okazaki fragment

Loop grows

Second Okazaki fragment nears completion

DNA polymerase III DNA polymerase I

3′ 5′ β clamp releases

3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase I attaches to remove the primer.

semidiscontinuous synthesis of DNA is illustrated in stages using the model from figure 14.18.

282 part III Genetic and Molecular Biology

3′ 5′

strand adds bases to the next Okazaki fragment.

3′ 5′

Figure 14.19 DNA synthesis by the replisome. The

New bases

5. After the β clamp is loaded, the DNA polymerase III on the lagging

occur 5′-to-3′ on both strands, with the complex moving to the left.

Lagging strand releases

Loop grows

3′ 5′

2. The “loop” in the lagging-strand template allows replication to

5′ 3′

3′ 5′

DNA polymerase I detaches after removing RNA primer

3′ 5′

replisome complex is held together by a number of proteins, including the clamp loader. As Pol III finishes an Okazaki fragment, the clamp loader loads a β subunit onto the next fragment, and transfers the Pol III to this new β subunit (figure 14.18). Current evidence also indicates that this replication complex is probably stationary, with the DNA strand moving through it like thread in a sewing machine, rather than the complex moving along the DNA strands. This stationary complex also pushes the newly synthesized DNA outward, which may aid in chromosome segregation. This process is summarized in figure 14.19.

Learning Outcomes Review 14.4

E. coli has three DNA polymerases: DNA Pol I, II, and III. Synthesis on one strand is discontinuous because DNA is antiparallel, and polymerases synthesize only in the 5′-to-3′ direction. Replication occurs at the replication fork, where the two strands are separated. Assembled here is a massive complex, the replisome, containing DNA polymerase III, primase, helicase, and other proteins. The lagging strand requires DNA polymerase I to remove the primers and replace them with DNA, and ligase to join Okazaki fragments. ■■

How does the replisome coordinate all of the necessary activities of DNA replication?

14.5

Eukaryotic Replication

Learning Outcomes 1. Compare eukaryotic replication with prokaryotic. 2. Explain the function of telomeres. 3. Evaluate the role of telomerase in cell division.

Eukaryotic replication is complicated by two main factors: the larger amount of DNA organized into multiple chromosomes, and the linear structure of the chromosomes. This process requires new enzymatic activities only for dealing with the ends of chromosomes; otherwise the basic enzymology is the same.

Eukaryotic replication uses multiple origins The sheer amount of DNA and how it is packaged constitute a problem for eukaryotes (figure 14.20). Eukaryotes usually have

multiple chromosomes that are each larger than the E. coli chromosome. If each chromosome had a single origin, the time necessary for replication would be prohibitive. Eukaryotic cells reduce the time needed for replication by using multiple origins for each chromosome, resulting in multiple replicons. The origins are not as sequence-specific as oriC, and their recognition seems to depend on chromatin structure as well as on sequence. The number of origins used can also be adjusted during the course of development, so that early on, when cell divisions need to be rapid, more origins are activated. Each origin must be used only once per cell cycle.

The eukaryotic replication fork is more complex Prior to S phase, helicase complexes are loaded onto possible replication origins, but not activated. Then, during S phase a subset of these are activated, and the rest of the replisome assembled. The action of the replisome differs from that in E. coli in a few ways we will highlight. Priming is accomplished by a complex of DNA polymerase α and primase. This complex synthesizes primers consisting of RNA and a short stretch of DNA. There are also different polymerases for the leading and lagging strands. DNA polymerase epsilon (Pol ε) is responsible for leading strand synthesis and DNA polymerase delta (Pol δ) does the lagging strand. The sliding clamp subunit that allows the enzyme complex to stay attached to the template is called PCNA (for proliferating cell nuclear antigen). The name comes from its identification as an antibody-inducing protein in proliferating (dividing) cells. The PCNA sliding clamp forms a trimer, but has similar structure and function to the β subunit sliding clamp. Synthesis on the lagging strand differs most from related events in prokaryotic DNA replication. The Okazaki fragments are significantly shorter: 200 bp instead of 1000 bp. As each fragment reaches the previous fragment, synthesis continues to displace the strand containing the previous primer. The displaced primer is removed by a nuclease, and the remaining fragments joined by ligase.

Archaeal and eukaryotic replication proteins are evolutionarily related

110,000×

Figure 14.20 DNA of a single human chromosome. 

This chromosome has been relieved of most of its packaging proteins, leaving the DNA in its native form. The residual protein scaffolding appears as the dark material in the lower part of the micrograph.  Don W. Fawcett/Science Source

The replisome, which can replicate both strands of DNA simultaneously, requires a variety of functions, including DNA unwinding (helicase), synthesis (primase and polymerase), and processivity factors (sliding clamps and their loaders). There is clear functional conservation of these activities across all domains of life, but major components appear to have evolved independently in bacteria, and in archaea/eukarya. Members of the DNA polymerase family of enzymes have similar structure and homologous synthetic domains. They are differentiated based on nonhomologous regions into seven subfamilies. The subfamily used by eukaryotes and archaea is the same, but different from the one used by bacteria. Replicative helicases all have a similar structure, forming a ring around a single strand of DNA, but the bacterial and archaeal/eukaryal enzymes are not homologous, and even move in the opposite direction along DNA. Lastly, the primase component of the replisome is also not homologous between bacteria and archaea/eukarya. chapter

14 DNA: The Genetic Material 283

The one function that is clearly homologous in all domains of life is the processivity factors: sliding clamps and clamp loaders.

Linear chromosomes have specialized ends The specialized structures found on the ends of eukaryotic chromo­ somes are called telomeres. These structures protect the ends of chromosomes from nucleases and maintain the integrity of linear chromosomes. These telomeres are composed of specific DNA sequences, but they are not made by the replication complex.

Replicating ends The very structure of a linear chromosome causes a cell problems in replicating the ends. The directionality of polymerases, combined with their requirement for a primer, creates this problem. Consider a simple linear molecule like the one in figure 14.21. Replication of one end of each template strand is simple, namely the 5′ end of the leading-strand template. When the polymerase reaches this end, synthesizing in the 5′-to-3′ direction, it eventually runs out of template and is finished. But on the other strand’s end, the 3′ end of the lagging strand, removal of the last primer on this end leaves a gap. This gap cannot be primed, meaning that the polymerase complex cannot finish this end properly. The result would be a gradual shortening of chromosomes with each round of cell division (figure 14.21).

The action of telomerase When the sequence of telomeres was determined, they were found to be composed of short repeated sequences of DNA. This

repeating nature is easily explained by their synthesis. They are made by an enzyme called telomerase, which uses an internal RNA as a template and not the DNA itself (figure 14.22). The use of the internal RNA template allows short stretches of DNA to be synthesized, composed of repeated nucleotide sequences complementary to the RNA of the enzyme. The other strand of these repeated units is synthesized by the usual action of the replication machinery copying the strand made by telomerase.

Telomerase, aging, and cancer A gradual shortening of the ends of chromosomes occurs in the absence of telomerase activity. During embryonic and childhood development in humans, telomerase activity is high, but it is low in most somatic cells of the adult. The exceptions are cells that must divide as part of their function, such as lymphocytes. The activity of telomerase in somatic cells is kept low by preventing the expression of the gene encoding this enzyme. To examine the role of telomerase, mice were produced that completely lacked telomerase activity. These mice appeared to be normal for up to six generations, but they showed steadily decreasing telomere length that eventually led to nonviable offspring. This implies a relationship between cell senescence (aging) and telomere length. Normal cells undergo only a specified number of divisions when grown in culture. This limit is at least partially based on telomere length. Support for the relationship between senescence and telomere length comes from experiments in which telomerase was introduced into fibroblasts in culture. These cells had their life span increased relative to controls that had no added telo­merase. Interestingly, these cells did not show the hallmarks of malignant

Replication first round 5′

3′

3′

5′ Leading strand (no problem)

5′

Lagging strand (problem at the end)

3′

3′ 5′

Last primer Origin Leading strand

Primer removal

3′ 5′

Lagging strand

5′ 3′ Replication second round

Figure 14.21 Replication of the end of linear DNA. 

Only one end is shown for simplicity; the problem exists at both ends. The leading strand can be completely replicated, but the lagging strand cannot be finished. When the last primer is removed, it cannot be replaced. During the next round of replication, when this shortened template is replicated, it will produce a shorter chromosome. 284 part III Genetic and Molecular Biology

Removed primer cannot be replaced

5′

3′

3′

5′

5′

3′

3′

Shortened template

5′

5′

T

T

14.6 DNA

G

3′

Repair

Synthesis by telomerase

Learning Outcomes

Telomerase 5′

T A

3′

T A

G C

G C

G C

G

T

C

A

T

A

Telomere extended by telomerase

G C

Template RNA is part of enzyme Telomerase moves and continues to extend telomere

5′

T

3′

T

G

G

G

Now ready to synthesize next repeat

G

T

T

G

A

A

C

T

G C

C

C

A

A

T

C

Figure 14.22 Action of telomerase. Telomerase contains an internal RNA that the enzyme uses as a template to extend the DNA of the chromosome end. Multiple rounds of synthesis by telomerase produce repeated sequences. This single strand is completed by normal synthesis using it as a template (not shown). cells, indicating that activation of telomerase alone does not make cells malignant. A relationship has been found, however, between tel­­o­me­­rase and cancer. Cancer cells do continue to divide indefinitely, and this would not be possible if their chromosomes were being continually shortened. Cancer cells generally show activation of telomerase, which allows them to maintain telomere length; but this is clearly only one aspect of conditions that allow them to escape normal growth controls.

?

Inquiry question How does the structure of eukaryotic

genomes affect replication? Does this introduce problems that are not faced by prokaryotes?

Learning Outcomes Review 14.5

Eukaryotic replication is complicated by a large amount of DNA organized into chromosomes, and by the linear nature of chromosomes. Eukaryotes replicate a large amount of DNA in a short time by using multiple origins of replication. Linear chromosomes end in telomeres, and the length of telomeres is correlated with the ability of cells to divide. The enzyme telomerase synthesizes the telomeres. Cancer cells show activation of telomerase, which extends the ability of the cells to divide. ■■

What might be the result of abnormal shortening of telomeres or a lack of telomerase activity?

1. Explain why DNA repair is critical for cells. 2. Describe the different forms of DNA repair.

Despite the high fidelity of DNA, replication errors still occur. With no way to correct errors, cells would accumulate an unacceptable level of deleterious or lethal mutations. A balance must exist between new variation arising by mutation, and the effects of deleterious mutations on the individual.

Cells are constantly exposed to DNA-damaging agents In addition to errors in DNA replication, cells are constantly exposed to DNA-damaging agents such as radiation (UV light and X-rays) and chemicals in the environment. Agents that damage DNA can lead to mutations, and any agent that increases the number of mutations above background levels is considered a mutagen. Sunlight itself includes UV radiation and is thus mutagenic. Ozone normally screens out much of the UV radiation in sunlight, but some remains. Regions in the southern hemisphere show increases in skin cancer that correlate with a seasonal “ozone hole,” indicating a relationship between sunlight and mutations. We are also exposed to mutagens in our diet, as either contaminant or natural plant products that are mutagenic. When a simple test to detect mutagens was devised, screening of possible sources indicated an amazing diversity of mutagens in the environment and in natural sources. As a result, consumer products are now screened to reduce the load of mutagens we are exposed to, but we cannot escape natural sources.

DNA repair restores damaged DNA The process that can restore damaged DNA to its native state is called DNA repair. One indication of the importance of DNA repair is the number of repair systems that have been characterized. All cells that have been examined show multiple pathways for repairing damaged DNA and for reversing errors that occur during replication. These systems help to reduce the mutational load on organisms to an acceptable level. The most common forms of repair appear to have evolved early in the history of cellular life, indicating their significance. DNA repair falls into two general categories: specific and nonspecific. Specific repair systems target a single kind of lesion in DNA and repair only that damage. Nonspecific forms of repair use a single mechanism to repair multiple kinds of lesions in DNA. The rest of this section will consider some specific examples of repair systems to illustrate the role of this process.

Mismatch repair Mutations due to DNA replication errors occur even less than predicted for a proofreading DNA polymerase. This is due to the action of mismatch repair (MMR), which removes incorrect bases that chapter

14 DNA: The Genetic Material 285

have been incorporated, replacing them with the correct base by copying the template strand. For this system to work, it must be able to distinguish between the template strand and the newly synthesized strand. In E. coli, this involves methylation of the base adenine in the sequence 5′ GATC 3′. This sequence is a palindrome, so the complement (3′ CTAG 5′) is the same 5′ to 3′, and the A in each strand is methylated. The methylase responsible does not act immediately after replication, leaving a short window for the MMR system to be able to identify the newly synthesized strand.

Photorepair When DNA is exposed to UV light, a number of photochemical reactions can occur. The most common produces a thymine dimer, where adjacent thymines become covalently linked together. There is a specific repair pathway, called photorepair, that recognizes this damage and reverses it. The enzyme photolyase binds to thymine DNA with adjacent thymines T A

T A

dimers, then uses energy from visible light to cleave the dimer and restore the thymine bases (figure 14.23). Genetic and biochemical analysis has revealed two distinct photolyase enzymes that recognize different photoproducts. These enzymes appear to be evolutionarily related to cryptochrome proteins that absorb visible light, but have no photolyase activity. In mammals, cryptochromes are part of the biochemical mechanism that links metabolic activities to daily light/dark cycles. Photorepair is found in a wide spectrum of organisms ranging from bacteria to animals. Early reports indicated that human skin was capable of photorepair, but later reports could find no activity. It now appears that placental mammals lack any photolyase.

Excision repair A common form of nonspecific repair is excision repair. In this pathway, a damaged region is removed, or excised, and is then replaced by DNA synthesis (figure 14.24). In E. coli, this action is accomplished by proteins encoded by the uvr A, B, and C genes. Although these genes were identified based on alleles that increased Damaged or incorrect base

UV light Helix distorted by thymine dimer

Thymine dimer Excision repair enzymes recognize damaged DNA T T

A

UvrABC complex binds damaged DNA

A

Photolyase binds to damaged DNA Excision of damaged strand Photolyase T T

A

A

Visible light Resynthesis by DNA polymerase Thymine dimer cleaved T A

DNA polymerase

T A

Figure 14.23 Repair of thymine dimer by photorepair. 

UV light can catalyze a photochemical reaction to form a covalent bond between two adjacent thymines, thereby creating a thymine dimer. A photolyase enzyme recognizes the damage and binds to the thymine dimer. The enzyme absorbs visible light and uses the energy to cleave the thymine dimer. 286 part III Genetic and Molecular Biology

Figure 14.24 Repair of damaged DNA by excision repair. 

Damaged DNA is recognized by the UvrABC complex, which binds to the damaged region and removes it. Synthesis by DNA polymerase replaces the damaged region. DNA ligase finishes the process (not shown).

sensitivity of the cell to UV light (hence the “uvr” in their names), the system can act on damage due to other mutagens. Excision repair follows three steps: (1) recognition of damage, (2) removal of the damaged region, and (3) resynthesis using the information on the undamaged strand as a template (figure 14.24). Recognition and excision are accomplished by the UvrABC complex. The UvrABC complex binds to damaged DNA and then cleaves a single strand on either side of the damage, removing it. In the synthesis stage, DNA Pol I or Pol II replaces the damaged DNA. This restores the original information in the damaged strand using the information in the complementary strand.

Other repair pathways Cells have other forms of both specific and nonspecific repair. Most of these are error-free but there is also an error-prone system. It may seem strange to have an error-prone pathway, but it can be thought of as a last-ditch effort to save a cell that has been exposed to such massive damage that it has overwhelmed the error-free systems. In fact, this system in E. coli is part of what is called the “SOS response.” Cells can also repair damage that produces breaks in DNA. These systems use enzymes related to those involved in recombination during meiosis (see chapter 11). It is thought

that recombination uses enzymes that originally evolved for DNA repair. The number of different systems, and the wide spectrum of damage that can be repaired, help to maintain the integrity of the genome. Accurate replication of the genome is useless if a cell cannot reverse errors that can occur during this process or repair damage due to environmental causes.

?

Inquiry question E. coli mutants with nonfunctional adenine methylase, deficient in adenine methylation (Dam) mutants, were isolated. What phenotype would you expect for Dam mutants?

Learning Outcomes Review 14.6

DNA repair is critical to remove replication errors, and to reverse the effects of DNA-damaging agents. Mismatch repair occurs immediately after replication to remove errors. Repair systems can be either specific or nonspecific. Photorepair is specific, removing thymine dimers caused by UV light. Excision repair is nonspecific, removing and replacing different kinds of damage. ■■

Could a cell survive with no form of DNA repair?

Chapter Review Molekuul/SPL/age fotostock

14.1 The Nature of the Genetic Material Griffith finds that bacterial cells can be transformed. Nonvirulent S. pneumoniae could take up an unknown substance from a virulent strain and become virulent. Avery, MacLeod, and McCarty identify the transforming principle. The transforming substance could be inactivated by DNA-digesting enzymes, but not by protein-digesting enzymes. Hershey and Chase demonstrate that phage genetic material is DNA. Radioactive labeling showed that the infectious agent of phage is its DNA, and not its protein.

14.2 DNA Structure DNA’s components were known, but its three-dimensional structure was a mystery. The nucleotide building blocks for DNA contain deoxyribose and the bases adenine (A), guanine (G), cytosine (C), and thymine (T). Phosphodiester bonds are formed between the 5′ phosphate of one nucleotide and the 3′ hydroxyl of another nucleotide (figure 14.4). Chargaff, Franklin, and Wilkins obtained some structural evidence. Chargaff found equal amounts of adenine and thymine, and of cytosine and guanine, in DNA. The bases exist primarily in keto and enol forms that exhibit hydrogen bonding. X-ray diffraction studies by Franklin and Wilkins indicated that DNA had a helical structure. The Watson–Crick model fits the available evidence (figures 14.8 and 14.9). DNA consists of two antiparallel polynucleotide strands wrapped about a common helical axis. These strands are held together by hydrogen

bonds forming specific base-pairs (A–T and G–C). The two strands are complementary; one strand can specify the other.

14.3 Basic Characteristics of DNA Replication Meselson and Stahl demonstrated the semiconservative mechanism (figure 14.11). Semiconservative replication uses each strand of a DNA molecule to specify the synthesis of a new strand. Meselson and Stahl showed this by using a heavy isotope of nitrogen and separating the replication products. Replication produces two new molecules each composed of one new strand and one old strand. DNA replication requires a template, nucleotides, and a polymerase enzyme. All new DNA molecules are produced by DNA polymerase copying a template. All known polymerases synthesize new DNA in the 5′‑to-3′ direction. These enzymes also require a primer. The building blocks used in replication are deoxynucleotide triphosphates with high-energy bonds; they do not require any additional energy.

14.4 Prokaryotic Replication Prokaryotic replication starts at a single origin. The E. coli origin has AT-rich sequences that are easily opened. The chromosome and its origin form a replicon. E. coli has at least three different DNA polymerases. Some DNA polymerases can also degrade DNA from one end, called exonuclease activity. Pol I, II, and III all have 3′-to-5′ exonuclease activity that can remove mispaired bases. Pol I can remove bases in the 5′-to-3′ direction, which is important to removing RNA primers. chapter

14 DNA: The Genetic Material 287

Unwinding DNA requires energy and causes torsional strain. DNA helicase uses energy from ATP to unwind DNA. The torsional strain introduced is removed by the enzyme DNA gyrase.

replication polymerase, which is a complex of two enzymes. The sliding clamp subunit was originally identified as protein produced by proliferating cells and is called PCNA.

Replication is semidiscontinuous. Replication is discontinuous on one strand (figure 14.15). The continuous strand is called the leading strand, and the discontinuous strand is called the lagging strand.

Archaeal and eukaryotic replication proteins are evolutionarily related. The replication proteins of archaea, including the sliding clamp, clamp loader, and DNA polymerases, are more similar to those of eukaryotes than to those of prokaryotes.

Synthesis occurs at the replication fork. The partial opening of a DNA strand forms two single-stranded regions called the replication fork. At the fork, synthesis on the leading strand requires a single primer, and the polymerase stays attached to the template because of the β subunit that acts as a sliding clamp. On the lagging strand, DNA primase adds primers periodically, and DNA Pol III synthesizes the Okazaki fragments. DNA Pol I removes primer segments, and DNA ligase joins the fragments.

Linear chromosomes have specialized ends. The ends of linear chromosomes are called telomeres. They are made by telomerase, not by the replication complex. Telomerase contains an internal RNA that acts as a template to extend the DNA of the chromosome end. Adult cells lack telomerase activity, and telomere shortening correlates with senescence.

The replisome contains all the necessary enzymes for replication. The replisome consists of two copies of Pol III, DNA primase, DNA helicase, and a number of accessory proteins. It moves in one direction by creating a loop in the lagging strand, allowing the antiparallel template strands to be copied in the same direction (figures 14.18 and 14.19).

Cells are constantly exposed to DNA-damaging agents. Errors from replication and damage induced by agents such as UV light and chemical mutagens can lead to mutations.

14.6 DNA Repair

DNA repair restores damaged DNA. Without repair mechanisms, cells would accumulate mutations until inviability occurred. Repair can be specific or nonspecific. Mismatch repair reverses replication errors using methylation to identify newly replicated DNA. Photorepair involves photolyase enzymes, which use visible light to reverse UV-induced damage. Two photolyases recognize specific kinds of DNA damage. Mammals appear to lack photolyase enzyme. Excision repair is nonspecific. Prokaryotic excision repair uses the uvr system, which can remove and replace a damaged region of DNA. Other repair pathways can repair breaks in DNA, and some are error prone.

14.5 Eukaryotic Replication Eukaryotic replication uses multiple origins. The sheer size and organization of eukaryotic chromosomes requires multiple origins of replication to be able to replicate DNA in the time available in S phase. The eukaryotic replication fork is more complex. The eukaryotic primase synthesizes a short stretch of RNA and then switches to making DNA. This primer is extended by the main

Visual Summary Genetic material

Nucleotides

Semiconservative

Phosphate

Double helix

Watson & Crick model

Antiparallel strands

Phosphodiester backbone Complementary bases

Base-pairing

single

Origin of replication

many

simple

Replication fork

complex

Primase

synthesis is

Helicase

Semidiscontinuous

DNA polymerases

held together by AT C≡G

DNA ligase

Mutagens

include

Prokaryotic replication

Nitrogenous base

described as

damage caused by

is

5-carbon sugar monomers

Components

DNA replication

DNA structure

DNA repair

if damaged

Eukaryotic replication

Telomerase

Leading strand

utilizes contains

Replisome

Lagging strand

Okazaki fragments

288 part III Genetic and Molecular Biology

Review Questions Molekuul/SPL/age fotostock

U N D E R S TA N D 1. What was the key finding from Griffith’s experiments using live and heat-killed pathogenic bacteria? a. b. c. d.

Bacteria with a smooth coat could kill mice. Bacteria with a rough coat are not lethal. DNA is the genetic material. Genetic material can be transferred from dead to live bacteria.

2. Which of the following is NOT a component of DNA? a. The pyrimidine uracil b. 5-carbon sugars

c. The purine adenine d. Phosphate groups

3. Chargaff studied the composition of DNA from different sources and found that a. b. c. d.

the number of phosphate groups always equals the number of 5-carbon sugars. the proportion of A equals that of C and the proportion of G equals that of T. the proportion of A equals that of T and the proportion of G equals that of C. purines bind to pyrimidines.

4. The bonds that hold two complementary strands of DNA together are a. hydrogen bonds. b. peptide bonds.

c. ionic bonds. d. phosphodiester bonds.

5. The basic mechanism of DNA replication is semiconservative with two new molecules, a. b. c. d.

each with new strands. one with all new strands and one with all old strands. each with one new and one old strand. each with a mixture of old and new strands.

6. One common feature of all DNA polymerases is that they a. b. c. d.

synthesize DNA in the 3′-to-5′ direction. synthesize DNA in the 5′-to-3′ direction. synthesize DNA in both directions by switching strands. do not require a primer.

7. Which of the following is NOT part of the Watson–Crick model of the structure of DNA? a. b. c. d.

DNA is composed of two strands. The two DNA strands are oriented in parallel (5′-to-3′). Purines bind to pyrimidines. DNA forms a double helix.

A P P LY 1. If one strand of a DNA is 5′ ATCGTTAAGCGAGTCA 3′, then the complementary strand would be: a. 5′ TAGCAATTCGCTCAGT 3′. b. 5′ ACTGAGCGAATTGCTA 3′. c. 5′ TGACTCGCTTAACGAT 3′. d. 5′ ATCGTTAAGCGAGTCA 3′. 2. Hershey and Chase used radioactive phosphorus and sulfur to a. label DNA and protein uniformly. b. differentially label DNA and protein. c. identify the transforming principle. d. Both b and c are correct. 3. The Meselson and Stahl experiment used a density label to be able to a. determine the directionality of DNA replication.

b. differentially label DNA and protein. c. distinguish between newly replicated and old strands. d. distinguish between replicated DNA and RNA primers. 4. The difference in leading- versus lagging-strand synthesis is a consequence of a. only the physical structure of DNA. b. only the activity of DNA polymerase enzymes. c. both the physical structure of DNA and the action of polymerase enzyme. d. the larger size of the lagging strand. 5. If the activity of DNA ligase were removed from replication, this would have a greater effect on a. synthesis on the lagging strand versus the leading strand. b. synthesis on the leading strand versus the lagging strand. c. priming of DNA synthesis versus actual DNA synthesis. d. photorepair of DNA versus DNA replication. 6. Successful DNA synthesis requires all of the following except a. helicase. c. DNA primase. b. endonuclease. d. DNA ligase. 7. The synthesis of telomeres a. uses DNA polymerase, but without the sliding clamp. b. uses enzymes involved in DNA repair. c. requires telomerase, which does not use a template. d. requires telomerase, which uses an internal RNA as a template. 8. When mutations that affected DNA replication were isolated, two kinds were found. In cultures that were not synchronized (that is, not all dividing at the same time), one class put an immediate halt to replication, whereas the other put a much slower stop to the process. The first class affects functions at the replication fork like polymerase and primase. The second class affects functions necessary for a. elongation on the lagging but not the leading strand. b. elongation on the leading but not the lagging strand. c. initiation: cells complete replication but cannot start a new round. d. the sliding clamp: loss makes the polymerase slower.

SYNTHESIZE 1. The work by Griffith provided the first indication that DNA was the genetic material. Review the four experiments outlined in figure 14.1. Predict the likely outcome for the following variations on this classic research. a. Heat-killed pathogenic and heat-killed nonpathogenic b. Heat-killed pathogenic and live nonpathogenic in the presence of an enzyme that digests proteins (proteases) c. Heat-killed pathogenic and live nonpathogenic in the presence of an enzyme that digests DNA (endonuclease) 2. In the Meselson–Stahl experiment, a control experiment was done to show that the hybrid bands after one round of replication were in fact two complete strands, one heavy and one light. Using the same experimental setup as detailed in the text, how can this be addressed? 3. Enzyme function is critically important for the proper replication of DNA. Predict the consequence of a loss of function for each of the following enzymes. a. DNA gyrase c. DNA ligase b. DNA polymerase III d. DNA polymerase I chapter

14 DNA: The Genetic Material 289

15 CHAPTER

Genes and How They Work Chapter Contents 15.1

The Nature of Genes

15.2

The Genetic Code

15.3

Prokaryotic Transcription

15.4

Eukaryotic Transcription

15.5

Eukaryotic pre-mRNA Splicing

15.6

The Structure of tRNA and Ribosomes

15.7

The Process of Translation

15.8

Summarizing Gene Expression

15.9

Mutation: Altered Genes

1 μm Dr. Gopal Murti/Science Source

Introduction

Visual Outline Gene expression

Prokaryotes Central dogma DNA→RNA→Protein

Coupled

Translation

Transcription

uses

RNA processing

Genetic code consists of

steps

64 codons

Initiation

requires

Eukaryotes

requires

RNA polymerase

Initiation

code for Amino acids

steps

Ribosome Stop

3′

Elongation

Elongation

E P A

Start

Termination

Termination 5′

You’ve seen how genes specify traits and how those traits can be followed in genetic crosses. You’ve also seen that the information in genes resides in the DNA molecule; the picture above shows all the DNA within the entire E. coli chromosome. Information in DNA is replicated by the cell and then partitioned equally during the process of cell division. The information in DNA is much like a blueprint for a building. The construction of the building uses the information in the blueprint, but requires building materials and carpenters and other skilled laborers using a variety of tools working together to actually construct the building. Similarly, the information in DNA requires nucleotide and amino acid building blocks, multiple forms of RNA, and many proteins acting in a coordinated fashion to make up the structure of a cell. We now turn to the nature of the genes themselves and how cells extract the information in DNA in the process of gene expression. Gene expression can be thought of as the conversion of genotype into the phenotype.

15.1 The

Experimental Procedure

Nature of Genes

No growth on minimal medium

Learning Outcomes 1. Evaluate the evidence for the one-gene/one-polypeptide hypothesis. 2. Distinguish between transcription and translation. 3. List the roles played by RNA in gene expression.

We know that DNA encodes proteins, but this knowledge alone tells us little about how the information in DNA can control cellular functions. Researchers had evidence that genetic mutations affected proteins, and in particular enzymes, long before the structure and code of DNA was known. In this section, we review the evidence of the link between genes and enzymes.

Garrod concluded that inherited disorders can involve specific enzymes In 1902, the British physician Archibald Garrod noted that certain diseases among his patients seemed to be more prevalent in particular families. By examining several generations of these families, he found that some diseases behaved as though caused by simple recessive alleles. Garrod concluded that these dis­­­­­­ orders were Mendelian traits, and that they had resulted from changes in the hereditary information in an ancestor of the affected families. Garrod investigated the disorder alkaptonuria in detail. Patients with alkaptonuria produce urine containing homogentisic acid (alkapton), which is rapidly oxidized in air, turning the urine black. In normal individuals, homogentisic acid is broken down into simpler substances. Garrod concluded that patients with alkaptonuria lack the enzyme necessary to catalyze this breakdown, further speculating that many other inherited diseases might also reflect enzyme deficiencies.

Beadle and Tatum showed that genes specify enzymes Garrod was actually ahead of his time as the connection between Mendelian alleles and enzymes was not codified for almost 40 years. In 1941 the link between traits followed in crosses and proteins was demonstrated by a series of experiments by George Beadle and Edward Tatum at Stanford University. They used the then new technology of inducing mutations by X-rays to collect a series of mutants for detailed study. Their work is summarized in figure 15.1, and described next.

Neurospora crassa, the bread mold Beadle and Tatum chose the bread mold Neurospora crassa for their experiments. This fungus can be grown readily in the laboratory on a defined medium consisting of only a carbon source (glucose), a vitamin (biotin), plus inorganic salts. This is called minimal medium because it represents the minimal requirements to support growth. Any cells that can grow on minimal medium must be able to synthesize all necessary biological molecules.

Growth on minimal medium plus arginine Wild-type Neurospora crassa

Mutagenize with X-rays

Grow on rich medium

arg mutants

Results Mutation in Enzyme

Plus Ornithine

Plus Citruline

Plus Arginosuccinate

Plus Arginine

E

F

G

H

Conclusion Glutamate Enzymes encoded by arg genes arg genes

Ornithine

Citruline

Arginine

Arginosuccinate

E

F

G

H

argE

argF

argG

argH

Figure 15.1 The Beadle and Tatum experiment. 

Wild-type Neurospora were mutagenized with X-rays to produce mutants deficient in the synthesis of arginine (top panel). The specific defect in each mutant was identified by growing on medium supplemented with intermediates in the biosynthetic pathway for arginine (middle panel). A mutant will grow only on media supplemented with an intermediate produced after the defective enzyme in the pathway for each mutant. The enzymes in the pathway can then be correlated with genes on chromosomes (bottom panel). chapter

15 Genes and How They Work 291

When Beadle and Tatum exposed spores to X-rays, they expected to find some mutants that would be unable to grow on minimal medium. These so-called nutritional mutants result from damage to genes encoding functions necessary to make important biological molecules.

Nutritional mutants To identify nutritional mutants, Beadle and Tatum first grew cultures on rich medium, then placed subcultures of individual fungal cells onto minimal medium. This identifies any cells that had lost the ability to make compounds necessary for growth. They concentrated on the ability to synthesize the amino acid arginine, where the biosynthetic pathway was known. To identify mutants unable to make arginine, they selected cultures that would grow only on minimal medium plus arginine. This resulted in a collection of independent mutants that were all unable to synthesize arginine, and that each could be genetically mapped to different chromosomal locations. This defined four genes they named argE, argF, argG, and argH.

One gene/one polypeptide With these tools in hand, Beadle and Tatum were able to genetically dissect the arginine biosynthetic pathway. By supplementing minimal medium with intermediates in the biochemical pathway, they could identify the specific lesion in each mutant. For any intermediate, if the mutation affects an enzyme that acts earlier in the pathway than the supplement, then growth should be supported—but not if the mutation affects a step after the intermediate used (figure 15.1). Using this approach, Beadle and Tatum were able to isolate a mutant strain defective for each enzyme in the biosynthetic pathway. Thus, each of the mutants they examined had a defect in a single enzyme, caused by a mutation at a single site on a chromosome. Beadle and Tatum concluded that genes specify the structure of enzymes, and that each gene encodes the structure of one enzyme (figure 15.1). They called this relationship the one-gene/oneenzyme hypothesis. Today, because many enzymes contain multiple polypeptide subunits, each encoded by a separate gene, the relationship is generalized to the one-gene/one-polypeptide hypothesis. This hypothesis clearly states the molecular relationship between genotype and phenotype. As you learn more about genomes and gene expression, you will see that this clear relationship is overly simplistic. Eukaryotic genes are more complex than those of prokaryotes, and some enzymes are composed, at least in part, of RNA, itself an intermediate in the production of proteins. Nevertheless, one gene/one polypeptide is a useful starting point for thinking about gene expression.

Data analysis If you made a double mutant with argE and

argG, which kind(s) of media would this strain grow on? In general, what can a double mutant tell you about the order of genes?

The central dogma describes information flow in cells as DNA to RNA to protein The conversion of genotype to phenotype requires information stored in DNA to be converted to protein. The nature of information flow in cells was first described by Francis Crick as the 292 part III Genetic and Molecular Biology

Prokaryotes Pro 5′ DNA C template strand

ary ryote otes t Eukaryotes

A 3′

G

C

T

T

T

Transcription

U

C

A

G

A

A

G

mRNA 3′

Translation

5′ Protein

Figure 15.2 The central dogma of molecular biology. 

DNA is transcribed to make mRNA, which is translated to make a protein.

central dogma of molecular biology. Information passes in one direction from the gene (DNA) to an RNA copy of the gene, and the RNA copy directs the sequential ­assembly of a chain of amino acids into a protein (figure 15.2). Stated briefly, DNA → RNA → protein The central dogma provides an intellectual framework that describes information flow in biological systems. We call the DNAto-RNA step transcription because it produces an exact copy of the DNA, much as a legal transcription contains the exact words of a court proceeding. The RNA-to-protein step is termed translation because it requires translating from the nucleic acid to the protein “languages.” Since the original formulation of the central dogma, a class of viruses called retroviruses was discovered that can convert their RNA genome into a DNA copy, using the viral enzyme reverse transcriptase. This conversion violates the direction of information flow of the central dogma, and the discovery forced an updating of the possible flow of information to include this “reverse” flow from RNA to DNA.

Replication DNA Transcription RNA

Reverse transcription Translation

Protein

Transcription makes an RNA copy of DNA The process of transcription produces an RNA copy of the information in DNA. That is, transcription is the DNA-directed synthesis of RNA by the enzyme RNA polymerase (figure 15.3). This process uses the principle of complementarity, described in chapter 14, to use DNA as a template to make RNA.

Translation uses information in RNA to synthesize proteins The process of translation is by necessity much more complex than transcription. In this case, RNA cannot be used as a direct template for a protein because there is no complementarity—that is, a sequence of amino acids cannot be aligned to an RNA template based on any kind of “chemical fit.” Molecular geneticists suggested that some kind of adapter molecule must exist that can interact with both RNA and amino acids, and transfer RNA (tRNA) was found to fill this role. This need for an intermediary adds a level of complexity to the process that is not seen in either DNA replication or transcription of RNA. Translation takes place on the ribosome, the cellular protein-synthesis machinery, and it requires the participation of multiple kinds of RNA and many proteins. Here we provide an outline of the processes; all are described in detail in the rest of the chapter.

RNA has multiple roles in gene expression 50 nm

Figure 15.3 RNA polymerase.  Platinum shadowed electron micrograph (freeze-fracture TEM) showing RNA polymerase molecules bound to DNA strands. Omikron/Science Source

Because DNA is double-stranded and RNA is singlestranded, only one of the two DNA strands needs to be copied. We call the strand that is copied the template strand. The RNA transcript’s sequence is complementary to the template strand. The strand of DNA not used as a template is called the coding strand. It has the same sequence as the RNA transcript, except that U (uracil) in the RNA is T (thymine) in the DNA-coding strand. Another naming convention for the two strands of the DNA is to call the coding strand the sense strand, as it has the same “sense” as the RNA. The template strand would then be the antisense strand: Coding (sense) 5′ –TCAGCCGTCAGCT– 3′ Template (antisense) 3′ –AGTCGGCAGTCGA– 5′

DNA

Transcription Coding 5′ –UCAGCCGUCAGCU– 3′ mRNA

The RNA transcript used to direct the synthesis of polypeptides is termed messenger RNA (mRNA). Its name reflects its use by the cell to carry the DNA message to the ribosome for processing.

?

Inquiry question RNA polymerase has no proofreading capacity. How does this affect the error rate in transcription compared with DNA replication? Why do you think it is more important for DNA polymerase than for RNA polymerase to proofread?

All RNAs are synthesized from a DNA template by transcription. Gene expression requires the participation of multiple kinds of RNA, each with different roles in the overall process. Here is a brief summary of these roles, which are described in detail in the remainder of the chapter: Messenger RNA. Even before the details of gene expression were unraveled, geneticists recognized that there must be an intermediate form of the information in DNA that can be transported out of the eukaryotic nucleus to the cytoplasm for ribosomal processing. This hypothesis was called the “messenger hypothesis,” and we retain this language in the name messenger RNA (mRNA). Ribosomal RNA. The class of RNA found in ribosomes is called ribosomal RNA (rRNA). There are multiple forms of rRNA, and rRNA is found in both ribosomal subunits. This rRNA is critical to the function of the ribosome. Transfer RNA. The intermediary adapter molecule between mRNA and amino acids is transfer RNA (tRNA). Transfer RNA molecules have amino acids covalently attached to one end and an anticodon that can base-pair with an mRNA codon at the other. The tRNAs act to interpret information in mRNA and to help position the amino acids on the ribosome. Small nuclear RNA. Small nuclear RNAs (snRNAs) are part of the machinery that is involved in nuclear processing of eukaryotic “pre-mRNA.” We discuss this splicing reaction later, in section 15.5. SRP RNA. In eukaryotes, where some proteins are synthesized by ribosomes on the rough endoplasmic reticulum (RER), this process is mediated by the signal recognition particle, or SRP, described in section 15.7. The SRP contains both RNA and proteins. Small RNAs. This class of RNA includes both microRNA (miRNA) and small interfering RNA (siRNA). These are involved in the control of gene expression discussed in chapter 16. chapter

15 Genes and How They Work 293

Learning Outcomes Review 15.1

Garrod showed that altered enzymes can cause metabolic disorders. Beadle and Tatum demonstrated that each gene encodes a unique enzyme. Genetic information flows from DNA (genes) to protein (enzymes) using messenger RNA as an intermediate. Transcription converts information in DNA into an RNA transcript, and translation converts this information into protein. RNA comes in several varieties having different functions; these include mRNA (the transcript), tRNA (the intermediary), and rRNA (in ribosomes), as well as snRNA, SNP RNA, and small RNAs (miRNA, siRNA). ■■

Why do cells need an adapter molecule like tRNA between RNA and protein?

information in DNA imply different methods of translating the information into protein. Sentence with Spaces WHY DID THE RED BAT EAT THE FAT RAT Delete one letter WHY DID HE RED BAT EAT THE FAT RAT Only one word changed Sentence with No Spaces WHY DIDTHE RED BAT EATTHEFAT RAT Delete one letter WHY DIDHER EDB ATEATTHEF ATRAT

15.2 The

Genetic Code

All words after deletion changed

Demonstrating the lack of spaces

Learning Outcomes 1. Summarize the experiments that revealed the genetic code. 2. Describe the characteristics of the genetic code. 3. Identify the relationship between codons and amino acids.

How does a sequence of nucleotides in a DNA molecule specify the sequence of amino acids in a polypeptide? The answer to this essential question came in 1961, through an experiment led by Francis Crick and Sydney Brenner. That experiment was so elegant and the result so critical to understanding the genetic code that we describe it here in detail.

The code is read in groups of three Crick and Brenner reasoned that the genetic code most likely consisted of a series of blocks of information called codons, each corresponding to an amino acid in the encoded protein. They further hypothesized that the information within one codon was probably a sequence of three nucleotides. With four DNA nucleotides (G, C, T, and A), using two in each codon can produce only 42, or 16, different codons—not enough to code for 20 amino acids. However, three nucleotides results in 43, or 64, different combinations of three, more than enough.

Do genetic messages include spaces? In theory, the sequence of codons in a gene could be punctuated with nucleotides between the codons that are not used, like the spaces that separate the words in this sentence. Alternatively, the codons could lie immediately adjacent to each other, forming a continuous sequence of nucleotides. If the information in the genetic message is separated by spaces, then altering any single word would not affect the entire sentence. In contrast, if all of the words are run together but read in groups of three, then any alteration that is not in groups of three would alter the entire sentence. These two ways of using 294 part III Genetic and Molecular Biology

To choose between these alternative mechanisms, Crick and his colleagues used a chemical to create mutations that caused singlebase insertions or deletions from a viral DNA molecule. They then showed that combining an insertion with a deletion restored function even though either one individually displayed loss of function. In this case, only the region between the insertion and the deletion would be altered. By choosing a region of the gene that encoded a part of the protein not critical to function, this small change did not cause a change in phenotype. When they combined a single deletion or two deletions near each other, the genetic message shifted, altering all of the amino acids after the deletion. When they made three deletions, however, the protein after the deletions was normal. They obtained the same results when they made additions to the DNA consisting of 1, 2, or 3 nt (nucleotides). Thus, Crick and Brenner concluded that the genetic code is read in increments of three nucleotides (in other words, it is a triplet code), and that reading occurs continuously without punctuation between the 3-nt units (figure 15.4). These experiments indicate the importance of the r­ eading frame for the genetic message. Because there is no punctuation, the reading frame established by the first codon in the sequence determines how all subsequent codons are read. We now call the kinds of mutations that Crick and Brenner used frameshift ­mutations because they alter the reading frame of the genetic message.

Nirenberg and others deciphered the code The determination of which of the 64 possible codons encoded each particular amino acid was one of the greatest triumphs of 20th-century biochemistry. Accomplishing this decryption depended on two related technologies: (1) cell-free biochemical systems that would support protein synthesis from a defined RNA, and (2) the ability to produce synthetic, defined RNAs that could be used in the cell-free system.

SCIENTIFIC THINKING Hypothesis: The genetic code is read in groups of three bases. Prediction: If the genetic code is read in groups of three, then deletion of one or two bases would shift the reading frame after the deletion. Deletion of three bases, however, would produce a protein with a single amino acid deleted but no change downstream. Test: Single-base deletion mutants are collected, each of which exhibits a mutant phenotype. Three of these deletions in a single region are combined to assess the effect of deletion of three bases.

The researchers then were able to use enzymes to synthesize defined 3-base sequences that could be tested for binding to the protein-synthesis machinery. This so-called triplet-binding assay allowed them to identify 54 of the 64 possible triplets. The organic chemist H. Gobind Khorana provided the final piece of the puzzle by using organic synthesis to produce artificial RNA molecules of defined sequence, and then examining what polypeptides they directed in cell-free systems. The combination of all of these methods allowed the determination of all 64 possible 3-nt sequences, and the full genetic code was determined (table 15.1).

One Base Deleted Met Pro Thr His Arg Asp Ala Ser

Amino acids

AUGCCUACGCACCGCGACGCAUCA Delete one base AUGCCUAGCACCGCGACGCAUCA Mett Pro Ser Thr Ala Thr His

All amino acids changed after deletion

Three Bases Deleted Met Pro Thr His Arg Asp Ala Ser

Amino acids

AUGCCUACGCACCGCGACGCAUCA Delete three bases AUGCCUCACCGCGACGCAUCA Mett Pro His Arg g Asp Ala Ser

Amino acids do not change after third deletion

Result: The combination of three deletions does not have the same drastic effect as the loss of one or two bases. Conclusion: The genetic code is read in groups of three. Further Experiments: If you also had mutants with single-base additions, what would be the effect of combining a deletion and an addition?

Figure 15.4 The genetic code is triplet. During a five-year period from 1961 to 1966, work performed primarily in American biochemist Marshall Nirenberg’s laboratory led to the elucidation of the genetic code. Nirenberg’s group first showed that adding the synthetic RNA molecule polyU (an RNA molecule consisting of a string of uracil nucleotides) to their cell-free systems produced the polypeptide polyphenylalanine (a string of phenylalanine amino acids). Therefore, UUU encodes phenylalanine. Next they used enzymes to produce RNA polymers with more than one nucleotide. These polymers allowed them to infer the composition of many of the possible codons, but not the order of bases in each codon.

The code is degenerate but specific Some obvious features of the code jump out of table 15.1. First, 61 of the 64 possible codons are used to specify amino acids. Three codons, UAA, UGA, and UAG, are reserved for another function: they signal “stop” and are known as stop codons. The only other form of “punctuation” in the code is that AUG is used to signal “start” and is therefore the start codon. In this case the codon has a dual function because it also encodes the amino acid methionine (Met). You can see that 61 codons are more than enough to encode 20 amino acids. That leaves lots of extra codons. One way to deal with this abundance would be to use only 20 of the 61 codons, but this is not what cells do. In reality, all 61 codons are used, making the code degenerate, which means that some amino acids are specified by more than one codon. The reverse, however, in which a single codon would specify more than one amino acid, is never found. This degeneracy is not uniform. Some amino acids have only one codon, and some have up to six. In addition, the degenerate base usually occurs in position 3 of a codon, such that the first two positions are the same, and two or four of the possible nucleotides at position 3 encode the same amino acid. (The nature of protein synthesis on ribosomes explains how this codon usage works, and it is discussed in section 15.7.)

The code is practically universal, but not quite The genetic code is the same in almost all organisms. The universality of the genetic code is among the strongest evidence that all living things share a common evolutionary heritage. Because the code is universal, genes can be transferred from one organism to another and can be successfully expressed in their new host (figure 15.5). This universality of gene expression is central to many of the advances of genetic engineering discussed in chapter 17. In 1979, investigators began to determine the complete nucleotide sequences of the mitochondrial genomes in humans, cattle, and mice. It came as something of a shock when these investigators learned that the genetic code used by these mammalian mitochondria was not quite the same as the “universal code” that has become so familiar to biologists. In the mitochondrial genomes, what should have been a stop codon, UGA, was instead read as the amino acid tryptophan; AUA was read as methionine rather than as isoleucine; and AGA and AGG were read as stop codons rather than as arginine. Furthermore, minor differences from the universal code have also been found in the genomes of chloroplasts and in ciliates (certain types of protists). chapter

15 Genes and How They Work 295

The Genetic Code

TA B L E 1 5 .1

SECOND LETTER

First Letter U

U UUU UUC UUA UUG

C

Phe Phenylalanine Leu Leucine

CUU CUC CUA

Leu Leucine

CUG A

AUU AUC

Ile

Isoleucine

AUA AUG G

GUA

UCU UCC UCA

Met Methionine; “Start”

Val Valine

GUG

A UAU

Ser Serine

G UGU

Tyr Tyrosine

UAC

UGC

UAA

“Stop”

UGA

UCG

UAG

“Stop”

UGG

CCU

CAU

CCC CCA

Pro Proline

CAA CAG

ACU ACC

AAU AAC

Thr Threonine

AAG

GCU

GAU

GCC GCA

Ala Alanine

GCG

CGC CGA

Gln Glutamine

AGU AGC AGA

Lys Lysine

AGG

GAC

GGC GGA

Glu Glutamate

GAG

Third Letter U C A G U

Arg Arginine

C A G

Ser Serine Arg Arginine

GGU

Asp Aspartate

GAA

“Stop” Trp Tryptophan

CGG

Asn Asparagine

AAA

ACG

Cys Cysteine

CGU

His Histidine

CAC

CCG

ACA

GUU GUC

C

U C A G U

Gly Glycine

GGG

C A G

A codon consists of three nucleotides read in the sequence shown. For example, ACU codes for threonine. The first letter, A, is in the First Letter column; the second letter, C, is in the Second Letter column; and the third letter, U, is in the Third Letter column. Each of the mRNA codons is recognized by a corresponding anticodon sequence on a tRNA molecule. Many amino acids are specified by more than one codon. For example, threonine is specified by four codons, which differ only in the third nucleotide (ACU, ACC, ACA, and ACG).

differently, particularly the portion associated with “stop” signals.

?

Inquiry question The genetic code is almost universal. Why do you think it is nearly universal?

Figure 15.6 Bacterial RNA polymerase and transcription initiation. a. RNA polymerase has two forms: core polymerase and

holoenzyme. b. The σ subunit of the holoenzyme recognizes promoter elements at –35 and –10 and binds to the DNA. The helix is opened at the –10 region, and transcription begins at the start site at +1. α α β β′ σ

Figure 15.5 Transgenic pig. The piglet on the right is a conventional piglet. The piglet on the left was engineered to express a gene from jellyfish that encodes green fluorescent protein. The color of this piglet’s nose is due to expression of this introduced gene. Such transgenic animals indicate the universal nature of the genetic code.

Core enzyme

Holoenzyme

Image courtesy of University of Missouri Extension

Thus, it appears that the genetic code is not quite universal. Some time ago, presumably after they began their endosymbiotic existence, mitochondria and chloroplasts began to read the code 296 part III Genetic and Molecular Biology

Prokaryotic RNA polymerase

a.

Learning Outcomes Review 15.2

The genetic code was shown to be nucleotide base triplets with two forms of punctuation and no spaces: three bases code for an amino acid, and the groups of three are read in order. Sixty-one codons specify amino acids, one of which also codes for “start,” and three codons indicate “stop,” for 64 total. Because some amino acids are specified by more than one codon, the code is termed degenerate. All codons encode only one amino acid, however. ■■

What would be the outcome if a codon specified more than one amino acid?

15.3 Prokaryotic

Initiation occurs at promoters

Transcription

Learning Outcomes 1. Describe the transcription process in bacteria. 2. Differentiate among initiation, elongation, and termination of transcription. 3. Define the unique features of prokaryotic transcription.

We begin an examination of gene expression by describing the process of transcription in prokaryotes. The description of eukaryotic transcription in section 15.4 will concentrate on their differences from prokaryotes.

Prokaryotes have a single RNA polymerase The single RNA polymerase of prokaryotes exists in two forms: the core polymerase and the holoenzyme. The core polymerase can synthesize RNA using a DNA template, but it cannot initiate synthesis accurately. The holoenzyme can accurately initiate synthesis. The core polymerase is composed of four subunits: two identical α subunits, a β subunit, and a β′ subunit (figure 15.6a). The

Coding strand

Accurate initiation of transcription requires two sites in DNA: one called a promoter that forms a recognition and binding site for the RNA polymerase, and the actual start site. The polymerase also needs a signal to end transcription, which we call a terminator. We then refer to the region from promoter to terminator as a transcription unit. The action of the polymerase moving along the DNA can be thought of as analogous to water flowing in a stream. We can speak of sites on the DNA as being “upstream” or “downstream” of the start site. We can also use this comparison to form a simple system for numbering bases in DNA to refer to positions in the transcription unit. The first base transcribed is called +1, and this numbering continues downstream until the last base is transcribed. Any bases upstream of the start site receive negative numbers, starting at –1. The promoter is a short sequence found upstream of the start site and is therefore not transcribed by the polymerase. Two 6-base sequences are common to bacterial promoters: one is located 35 nt upstream of the start site (–35), and the other is located 10 nt upstream of the start site (–10) (figure 15.6b). These two sites provide the promoter with asymmetry; they indicate not only the site of initiation, but also the direction of transcription. The binding of RNA polymerase to the promoter is the first step in transcription. Promoter binding is controlled by the σ subunit of the RNA polymerase holoenzyme, which recognizes the –35 sequence in the promoter and positions the RNA polymerase at the correct start site, oriented to transcribe in the correct direction.

RNA polymerase bound to unwound DNA

σ binds to DNA

TATAAT Promoter (−10 sequence) Template strand

two α subunits help to hold the complex together and can bind to regulatory molecules. The active site of the enzyme is formed by the β and β′ subunits, which bind to the DNA template and the ribonucleotide triphosphate precursors. The holoenzyme is formed by the addition of a σ (sigma) subunit to the core polymerase (figure 15.6a). Its ability to recognize specific signals in DNA allows RNA polymerase to locate the beginning of genes, which is critical to its function. Note that initiation of mRNA synthesis does not require a primer, in contrast to DNA replication.

5′

5′ Downstream 3′

Transcription bubble

3′

Start site (+1)

σ dissociates ATP Helix opens at −10 sequence

TTGACA Promoter (−35 sequence)

5′ 3′

Upstream

Start site RNA synthesis begins

5′ 3′

b. chapter

15 Genes and How They Work 297

Once bound to the promoter, the RNA polymerase begins to unwind the DNA helix at the –10 site (figure 15.6b). The polymerase covers a region of about 75 bp but unwinds only about 12 to 14 bp.

?

RNA polymerase DNA Start site

Inquiry question The prokaryotic promoter has two

distinct elements that are not identical. How is this important to the initiation of transcription? Unwinding

Coding strand

Elongation adds successive nucleotides In prokaryotes, the transcription of the RNA chain usually starts with ATP or GTP. One of these forms the 5′ end of the chain, which grows 5′-to-3′ as ribonucleotides are added to the 3′ end. As the RNA polymerase molecule leaves the promoter region, the σ factor is no longer required, although it may remain in association with the enzyme. This process of leaving the promoter, called clearance, or escape, involves more than just synthesizing the first few nucleo­ tides of the transcript and moving on. The enzyme made strong contacts with the DNA of the promoter region during initiation, and it is necessary to break these contacts to be able to move progressively down the template. The enzyme goes through conformational changes during this clearance stage, and subsequently contacts less of the DNA than it does during the initial promoter binding. The region containing the RNA polymerase, the DNA template, and the growing RNA transcript is called the transcription bubble because it contains a locally unwound “bubble” of DNA (figure 15.7). The polymerase makes a variety of contacts with DNA, including about 18 bp downstream of the active site. The bubble itself is 12–14 nt, including an 8–10 bp RNA:DNA hybrid. Finally about 5 nt of the RNA contact the enzyme in an exit channel. The transcription bubble created by RNA polymerase moves down the bacterial DNA at a constant rate, about 50 nt/sec, with the growing RNA moving out the exit channel. As the enzyme moves along the DNA, the DNA in front of the enzyme is opened, and the now-transcribed DNA is rewound as it leaves the bubble.

Termination occurs at specific sites The end of a bacterial transcription unit is marked by terminator sequences that signal “stop” to the polymerase. Reaching these sequences causes the formation of phosphodiester bonds to cease, the RNA–DNA hybrid within the transcription bubble to dissociate, the RNA polymerase to release the DNA, and the DNA within the transcription bubble to rewind. The simplest terminators consist of a series of G–C basepairs followed by a series of A–T base-pairs. The RNA transcript of this stop region can form a double-stranded structure in the GC region called a hairpin, which is followed by four or more uracil (U) ribonucleotides (figure 15.8). Formation of the hairpin causes the RNA polymerase to pause, placing it directly over the run of four uracils. The pairing of U with the DNA’s A is the weakest of the four hybrid base-pairs, and it is not strong 298 part III Genetic and Molecular Biology

Rewinding

3′ 5′

3′

5′

Downstream

3′ Upstream

Template strand

mRNA

Transcription bubble

5′

Figure 15.7 Model of a transcription bubble. The DNA

duplex is unwound by the RNA polymerase complex, rewinding at the end of the bubble. One of the strands of DNA functions as a template, and nucleotide building blocks are added to the 3′ end of the growing RNA. There is a short region of RNA–DNA hybrid within the bubble.

RNA polymerase DNA

DNA and RNA polymerase dissociates

mRNA dissociates from DNA

3′ 5′ 5′ 3′

Four or more U ribonucleotides

mRNA hairpin causes RNA polymerase to pause

5′

Cytosine Guanine Adenine Uracil

Figure 15.8 Bacterial transcription terminator. The

self-complementary G–C region forms a double-stranded stem with a single-stranded loop called a hairpin. The stretch of U’s forms a less stable RNA–DNA hybrid that falls off the enzyme.

enough to hold the hybrid strands when the polymerase pauses. Instead, the RNA strand dissociates from the DNA within the transcription bubble, and transcription stops. A variety of protein factors also act at these terminators to aid in terminating transcription.

Prokaryotic transcription is coupled to translation In prokaryotes, the mRNA produced by transcription begins to be translated before transcription is finished—that is, transcription and translation are coupled (figure 15.9). As soon as a 5′ end of the mRNA becomes available, ribosomes are loaded onto this to begin translation. In eukaryotes where transcription occurs in the nucleus and is followed by translation in the cytoplasm, this kind of coupling does not occur. Another difference between prokaryotic and eukaryotic gene expression is that the mRNA produced in prokaryotes may contain multiple genes. Prokaryotic genes are often organized such that genes encoding related functions are clustered together. This grouping of functionally related genes is referred to as an operon. An operon is a single transcription unit that encodes multiple enzymes necessary for a biochemical pathway. When genes are clustered by function, they can be regulated together, a topic that we return to in chapter 16.

Learning Outcomes Review 15.3

Transcription in bacteria is accomplished by RNA polymerase, which has two forms: a core polymerase and a holoenzyme. Initiation is accomplished by the holoenzyme form, which can accurately recognize promoter sequences. Elongation consists of RNA synthesis by the core enzyme, which adds RNA nucleotides in sequence until it reaches a terminator where synthesis stops; then the transcript is released. In prokaryotes, translation of the RNA transcript begins before transcription is finished, making the processes coupled. ■■

Yeast are unicellular organisms like bacteria; would you expect them to have the same transcription/translation coupling?

15.4 Eukaryotic

Transcription

Learning Outcomes 1. Differentiate promoters for the three eukaryotic polymerases. 2. Describe the processing of eukaryotic transcripts. 3. Explain the differences between bacterial and eukaryotic elongation of transcription.

The basic mechanism of transcription by RNA polymerase is similar in all systems. In fact, the RNA polymerases of bacteria, archaea, and eukaryotes all appear to be descended from the same ancestral enzyme. However, the details of RNA production in eukaryotes differ enough to merit consideration.

Eukaryotes have three RNA polymerases

0.25 μm RNA polymerase

mRNA Ribosomes

DNA

Polyribosome Polypeptide chains

Figure 15.9 Transcription and translation are coupled in prokaryotes. In this micrograph of gene expression in E. coli, translation is occurring during transcription. The arrows point to RNA polymerase enzymes, and ribosomes are attached to the mRNAs extending from the polymerase. Polypeptides being synthesized by ribosomes, which are not visible in the micrograph, have been added to the last mRNA in the drawing. (top): Professor

Oscar Miller/Science Source

An obvious difference between bacteria and eukaryotes is that bacteria have a single RNA polymerase, while eukaryotes have three distinct RNA polymerases, with different cellular roles. The enzyme RNA polymerase I transcribes rRNA, RNA polymerase II transcribes mRNA and some small nuclear RNAs, and RNA polymerase III transcribes tRNA and some other small RNAs. Together, these three enzymes accomplish all transcription in eukaryotic cells. The three eukaryotic RNA polymerases require different control elements in the DNA to allow each polymerase to recognize where to initiate transcription. Each polymerase recognizes a different promoter structure.

RNA polymerase I promoters RNA polymerase I promoters at first puzzled biologists, because comparisons of rRNA genes between species showed no similarities outside the coding region. The current view is that these promoters are specific for each species, which explains the lack of conserved sequences in cross-species comparisons.

RNA polymerase II promoters Most of the first eukaryotic genes cloned contained a sequence, TATA, upstream of the start site. The similarity of the TATA sequence to the prokaryotic -10 element led to the idea that this was chapter

15 Genes and How They Work 299

the promoter. This has proved too simple and has been replaced by the idea of a “core promoter” that can be composed of a number of distinct elements, including the TATA box.

RNA polymerase III promoters Promoters for RNA polymerase III also were a source of surprise for biologists examining the control of eukaryotic gene expression. A common technique for analyzing regulatory regions was to make successive deletions from the 5′ end of genes until enough was deleted to abolish specific transcription. In the case of tRNA genes, 5′ deletions had no effect on expression! The promoters were found within the actual gene itself. This has not proved to be the case for all polymerase III genes, but appears to be for most.

The core promoter binds factors that recruit RNA Pol II The initiation at RNA polymerase II promoters is analogous to prokaryotic initiation, but instead of a single factor allowing promoter recognition, eukaryotes use a host of transcription factors. These factors act cooperatively to recruit RNA polymerase II to a promoter. The transcription factors interact with RNA polymerase II to form an initiation complex at the promoter (figure 15.10). We explore this complex in detail in chapter 16 when we describe the control of gene expression. When the transcript reaches about 20 nucleotides, it is modified by the addition of GTP to the 5′ PO4– group, forming what is known as the 5′ cap (figure 15.11). This cap is joined to the transcript by its 5′ end—the only 5′-to-5′ bond found in nucleic acids. The G in the GTP is also modified by the addition of a methyl

Other transcription factors

group, so it is often called a methyl-G cap. This structure is important for translation, RNA stability, and further processing. Another event that can occur during early elongation is that the Pol may pause. This usually occurs within 50 bp of initiation, and now appears to be a common phenomenon. Initially described for heat shock genes, where it allows the rapid induction of transcription upon heat shock, global screens of promoter occupancy indicate that a majority of genes in multicellular organisms may harbor a paused polymerase. These data are difficult to interpret as they cannot distinguish between abortive short transcripts and a paused Pol that can restart. For the latter, a complex interplay of promoter elements, elongation factors, nucleosome occupancy, and even the capping enzyme all participate. The integration of the control of transcript initiation and that of release from promoter proximal pausing is not clear at this point.

The transcription elongation complex can recruit other factors The carboxy terminal domain (CTD) of the largest subunit of RNA polymerase II is important to recruit other factors that with the polymerase make up the transcription elongation complex. The CTD consists of 7 amino acids repeated many times (52 repeats in humans). The CTD contains serines that can be phosphorylated, which then interact with factors involved in elongation and RNA-modifying enzymes. For example, the capping enzyme just described interacts with the CTD soon after initiation. The elongation factors that interact with the CTD include ones that are involved in the release of pausing, and in progressing through nucleosomes. Early in vitro studies indicated that RNA

RNA polymerase II

Eukaryotic DNA

Transcription factor

Initiation complex

TATA box 1. A transcription factor recognizes and binds to the TATA box sequence, which is part of the core promoter.

2. Other transcription factors are recruited, and the initiation complex begins to build.

3. Ultimately, RNA polymerase II associates with the transcription factors and the DNA, forming the initiation complex, and transcription begins.

Figure 15.10 Eukaryotic initiation complex. Unlike transcription in prokaryotic cells, in which the RNA polymerase recognizes and binds to the promoter, eukaryotic transcription requires the binding of transcription factors to the promoter before RNA polymerase II binds to the DNA. The association of transcription factors and RNA polymerase II at the promoter is called the initiation complex. 300 part III Genetic and Molecular Biology

Figure 15.11  Posttranscriptional modifications to 5′ and 3′ ends.  Eukaryotic mRNA

molecules are modified in the nucleus with the addition of a methylated GTP to the 5′ end of the transcript, called the 5′ cap, and a long chain of adenine residues to the 3′ end of the transcript, called the 3′ poly-A tail.

5′ cap HO

OH

CH2

N+ CH3

P

P

P

y-A

ol 3′ p

+ Methyl group mRNA

P P P G 5′

AAUAAA

AA

tail

AA

A

3′

AA

CH3

Pol cannot transcribe DNA that is assembled into chromatin. Clearly this is not the case in vivo, and this is due to elongation factors that interact with the Pol, and with nucleosomes to result in removal of nucleosomes and repositioning behind the polymerase. These factors include FACT (facilitates chromatin transcription), factors that modify histones, and chromatin-remodeling complexes (discussed in chapter 16).

Polyadenylation is involved in terminating mRNA transcripts A distinctive feature of eukaryotic transcription is that the 3′ end of an mRNA is not the end of the transcript. The transcript is cleaved downstream of a specific site (AAUAAA) while the polymerase is elongating and a series of 100 to 200 adenosine residues, the 3′ poly-A tail, is added to the mRNA. The enzyme responsible for this is poly-A polymerase (figure 15.11). A complex of factors, including Poly-A polymerase, associates with the elongating polymerase via the phosphorylated CTD. The actual mechanism of transcription termination is not clear, but it involves the polymerase pausing around the poly site, and while the free mRNA is being polyadenylated, the remaining transcript is progressively removed by an exonuclease and the elongation complex dissociated. Termination of transcripts that are not polyadenlyated uses a mechanism that requires other factors. The one common feature to transcription termination in all systems is something that causes RNA polymerase to pause so that the elongation complex can be disassembled.

Primary transcripts are modified to produce mRNAs It is clear from the preceding discussion that an mRNA in eukaryotes is not the same as the primary transcript of a gene. With the modified 5′ end, a 3′ end produced by another enzyme, and the removal of introns, the mRNA is an extensively modified form of the primary transcript. The greatest modification to the primary transcript is the removal of noncoding sequences, which allows greater flexibility for eukaryotic gene expression. This process of pre-mRNA splicing is accomplished by a macromolecular machine called the spliceosome. We will now consider this complex topic and some of its implications.

Learning Outcomes Review 15.4

Eukaryotes have three RNA polymerases called polymerase I, II, and III. Each synthesizes a different RNA and recognizes its own promoter. The RNA polymerase I promoter is species-specific. The polymerase II core promoter consists of multiple elements. The polymerase III promoter is internal to the gene. Polymerase II is responsible for mRNA synthesis. Elongation by Pol II requires different factors, and modifying enzymes act during transcription to cap, polyadenylate, and splice the transcript. ■■

Does the complexity of the eukaryotic genome require three polymerases?

15.5 Eukaryotic

Splicing

pre-mRNA

Learning Outcomes 1. Explain the relationship between genes and proteins in prokaryotes and eukaryotes. 2. Describe the splicing reaction for pre-mRNA. 3. Illustrate how splicing changes the nature of genes.

The first genes isolated were prokaryotic genes found in E.  coli and its viruses. A clear picture of the nature of gene expression emerged from these systems before any eukaryotic genes were isolated. It was assumed that although details would differ, the outline of gene expression in eukaryotes would be similar. As we have seen in the previous section, this proved to be wrong.

Eukaryotic genes may contain interruptions Many eukaryotic genes were found to contain sequences that were not represented in the mRNA. It is hard to exaggerate how unexpected this finding was. A basic tenet of molecular biology based on E. coli was that a gene was colinear with its protein product— that is, the sequence of bases in the gene corresponds to the chapter

15 Genes and How They Work 301

E1

I1

E2

I2

E3

I3

DNA template

E4

The splicing reaction

I4

Exons Introns

Transcription

3′ poly-A tail

5′ cap Primary RNA transcript Introns are removed 3′ poly-A tail 5′ cap

a.

Mature mRNA Intron 1

4 DNA 5

b.

mRNA 3

2 6

7 Exon

c.

Figure 15.12 Eukaryotic genes contain introns and exons. a. Eukaryotic genes contain sequences that form the coding

sequence called exons and intervening sequences called introns.

b. An electron micrograph showing hybrids formed with the mRNA

and the DNA of the ovalbumin gene, which has seven introns. Introns within the DNA sequence have no corresponding sequence in the mRNA and thus appear as seven loops. c. A schematic drawing of the micrograph. (b): Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

Data analysis Illustrate how the gene in figure 15.12

could encode multiple transcripts.

sequence of bases in the mRNA, which in turn corresponds to the sequence of amino acids in the protein. In the case of eukaryotes, genes can be interrupted by sequences that are not represented in the mRNA and the protein. The term “split genes” was used at the time, but the nomenclature that has stuck describes the unexpected nature of these sequences. We call the noncoding DNA that interrupts the sequence of the gene “intervening sequences,” or introns, and we call the coding sequences exons because they are expressed (figure 15.12).

The spliceosome is a splicing machine It is still true that the mature eukaryotic mRNA is colinear with its protein product, but a gene that contains introns is not. Imagine looking at an interstate highway from a satellite. Scattered randomly along the thread of concrete would be cars, some moving in clusters, others individually; most of the road would be bare. That is what a eukaryotic gene is like—scattered exons embedded within much longer sequences of introns. In humans, only 1 to 1.5% of the genome is devoted to the exons that encode proteins; 24% is devoted to the noncoding introns within which these exons are embedded. 302 part III Genetic and Molecular Biology

The obvious question is—How do eukaryotic cells deal with the noncoding introns? The answer is that the primary transcript is cut and put back together to produce the mature mRNA. The latter process is referred to as pre-mRNA splicing, and it occurs in the nucleus prior to the export of the mRNA to the cytoplasm. The intron–exon junctions are recognized by small ­nuclear ribonucleoprotein particles, called snRNPs (pronounced “snurps”). The snRNPs are complexes composed of snRNA and protein. These snRNPs then cluster together with other associated proteins to form a larger complex called the spliceosome, which is responsible for the splicing, or removal, of the introns. For splicing to occur accurately, the spliceosome must be able to recognize intron–exon junctions. Introns all begin with the same 2-base sequence and end with another 2-base sequence that tags them for removal. In addition, within the intron there is a conserved A nucleotide, called the branch point, which is important for the splicing reaction (figure 15.13). The splicing process begins with cleavage of the 5′ end of the intron. This 5′ end becomes attached to the 2′ OH of the branch point A, forming a branched structure called a lariat due to its resemblance to a cowboy’s lariat in a rope (figure 15.13). The 3′ end of the first exon is then used to displace the 3′ end of the intron, joining the two exons together and releasing the intron as a lariat. The splicing process takes place during transcription. The transcript exits the enzyme near the CTD, allowing recruitment of splicing factors and the spliceosomal RNPs. There is evidence that the rate of transcription, which is not constant, can affect splice site selection. Pausing of the enzyme and the formation of secondary structure in the transcript can make splice sites inaccessible.

Distribution of introns No rules govern the number of introns per gene or the sizes of introns and exons. Some genes have no introns; others have as many as 50. The sizes of exons range from a few nucleotides to 7500 nt, and the sizes of introns are equally variable. The presence of introns partly explains why so little of a eukaryotic genome is actually composed of “coding sequences” (see chapter 18 for results from the Human Genome Project). One explanation for the existence of introns suggests that exons represent functional domains of proteins, and that the intron– exon arrangements found in genes represent the shuffling of these functional units over long periods of evolutionary time. This hypothesis, called exon shuffling, was proposed soon after the discovery of introns and has been the subject of much debate over the years. The recent flood of genomic data has shed light on this issue by allowing statistical analysis of the placement of introns and on intron–exon structure. This analysis has provided­support for the exon shuffling hypothesis for many genes; however, it is also clearly not universal, because all proteins do not show this kind of pattern. It is possible that introns do not have a single origin, and therefore cannot be explained by a single hypothesis.

Splicing can produce multiple transcripts from the same gene We call all of the RNAs produced from a genome the transcriptome, and all the proteins produced from a genome the proteome.

snRNA Exon 1

snRNPs Intron

5′

Exon 2

A

3′

Branch point A

1. snRNA forms base-pairs with 5′ end of intron, and at branch site.

A

Spliceosome

5′

3′

2. snRNPs associate with other factors to form spliceosome.

Lariat A

5′

3′

3. 5′ end of intron is removed and forms bond at branch site, forming a lariat. The 3′ end of the intron is then cut.

Exon 1 5′

Excised intron

Exon 2

Mature mRNA

3′

4. Exons are joined; spliceosome disassembles.

High-throughput systems to sequence cellular RNAs have produced a flood of data on patterns of transcription. More than 200,000 transcripts have been detected, leading to estimates of up to 95% of human genes producing multiple splice products. Estimates of the average number of transcripts per gene vary widely from as many as 10 to as few as 1.5 per gene. A conservative estimate from genome databases provides an estimate of 85,000 possible protein-encoding transcripts. Unfortunately, only a minority of these are experimentally verified. The situation is further clouded by the observation that as many as 80% of protein-coding genes appear to have one predominant transcript. The predominant transcript and minor transcripts can switch with different cell and tissue types, while the overall level of transcription remains the same. While there are well-characterized examples of alternative splicing producing different functional proteins, the overall biological significance remains unclear. The importance of a normal splicing pattern is also suggested by the observation that human genetic disorders can be due to aberrant splicing. It is clear that the transcriptome is much more diverse than one gene equals one transcript, but it is not clear the extent to which alternative splicing contributes to proteome diversity. The actual size of the proteome is also not yet clear, although progress is being made using mass spectrometry (see chapter 18).

Learning Outcomes Review 15.5

Prokaryotic genes are colinear with their protein products. Eukaryotic genes, by contrast, have expressed regions called exons, and sequences that interrupt the exons called introns. Introns are removed, and exons joined together, by the spliceosome. Alternative splicing incorporates different exons to generate different mRNAs, and thus different proteins, from the same gene. This appears to be widespread in multicellular organisms. ■■

What advantages would alternative splicing confer on an organism?

Figure 15.13 Pre-mRNA splicing by the spliceosome. 

Particles called snRNPs contain snRNA that interacts with the 5′ end of an intron and with a branch site internal to the intron. Several snRNPs come together with other proteins to form the spliceosome. As the intron forms a loop, the 5′ end is cut and linked to a site near the 3′ end of the intron. The intron forms a lariat that is excised, and the exons are spliced together. The spliceosome then disassembles and releases the spliced mRNA.

If every gene was spliced to include all exons, then the number of genes, transcripts, and proteins would all be equal. Unfortunately, it is not so simple: a single primary transcript can be spliced into different mRNAs by using different sets of exons, a process called alternative splicing. Before the human genome project (see chapter 18), it was estimated that humans had 100,000 genes. The current number is down to around 20,000 genes that encode proteins, but more than 80,000 protein encoding transcripts have been identified. Alternative splicing provides a way out of this conundrum.

15.6 The

Structure of tRNA and Ribosomes

Learning Outcomes 1. Explain why the tRNA charging reaction is critical to translation. 2. Identify the tRNA-binding sites in the ribosome.

The ribosome is the key macromolecular machine involved in translation, but it also requires the participation of mRNA, tRNA, and a host of other factors. Critical to this process is the interaction of the ribosomes with tRNA and mRNA. To understand this, we first examine the structure of the tRNA adapter molecule and the ribosome itself. chapter

15 Genes and How They Work 303

Aminoacyl-tRNA synthetases attach amino acids to tRNA Each amino acid must be attached to a tRNA with the correct anticodon for protein synthesis to proceed. This covalent attachment is accomplished by the action of activating enzymes called aminoacyl-tRNA synthetases. One of these enzymes is present for each of the 20 common amino acids.

tRNA structure Transfer RNA is a bifunctional molecule that must be able to interact with mRNA and with amino acids. The structure of tRNAs is highly conserved in all living systems, and it can be formed into a cloverleaf type of structure based on intramolecular base-pairing that produces double-stranded regions. This primary structure is then folded in space to form an L-shaped molecule that has two functional ends: the acceptor stem and the anticodon loop (figure 15.14). The acceptor stem is the 3′ end of the molecule, which always ends in 5′ CCA 3′. The amino acid is attached to this end of the molecule. The anticodon loop is the bottom loop of the cloverleaf, and it can base-pair with codons in mRNA.

The charging reaction The aminoacyl-tRNA synthetases must be able to recognize specific tRNA molecules as well as their corresponding amino acids. Although 61 codons code for amino acids, there are actually not 61 tRNAs in cells, although the number varies from species to species. Therefore, some aminoacyl-tRNA synthetases must be able to recognize more than one tRNA—but each recognizes only a single amino acid. The reaction catalyzed by the enzymes is called the tRNA charging reaction, and the product is an amino acid joined to a tRNA, now called a charged tRNA. An ATP molecule provides energy for this endergonic reaction. The charged tRNA produced by the reaction is an activated intermediate that can undergo the peptide bond-forming reaction without an additional input of energy. The charging reaction joins the acceptor stem to the carboxyl terminus of an amino acid (figure 15.15). Keeping this directionality in mind is critical to understanding the function of the ribosome, 2D “Cloverleaf” Model 3′

Acceptor end

3D Ribbon-Like Model Acceptor end

because each peptide bond will be formed between the amino group of one amino acid and the carboxyl group of another amino acid. The correct attachment of amino acids to tRNAs is important because the ribosome does not verify this attachment. Ribosomes can ensure only that the codon–anticodon pairing is correct. In an elegant experiment, cysteine was converted chemically to alanine after the charging reaction, when the amino acid was already attached to tRNA. When this charged tRNA was used in an in vitro protein synthesis system, alanine was incorporated in the place of cysteine, showing that the ribosome cannot “proofread” the amino acids attached to tRNA. In a very real sense, therefore, the charging reaction is the actual translation step; amino acids are incorporated into a peptide based solely on the tRNA anticodon and its interaction with the mRNA.

The ribosome has multiple tRNA-binding sites The synthesis of any biopolymer can be broken down into initiation, elongation, and termination—you have seen this division for DNA replication as well as for transcription. In the case of translation, or protein synthesis, all three of these steps take place on the ribosome, a large macromolecular assembly consisting of rRNA and proteins. Details of the process by which the two ribosome subunits are assembled during initiation are described shortly. For the ribosome to function it must be able to bind to at least two charged tRNAs at once so that a peptide bond can be formed between their amino acids, as described in the overview in section 15.1. The bacterial ribosome contains three binding sites, summarized in figure 15.16: ■■

■■

■■

The A site (aminoacyl) binds to the tRNA carrying the next amino acid to be added. The P site (peptidyl) binds to the tRNA attached to the growing peptide chain. The E site (exit) binds the tRNA that carried the previous amino acid added.

Transfer RNAs move through these sites successively during the process of elongation. Relative to the mRNA, the sites are 3D Space-Filled Model Acceptor end

Icon Acceptor end

5′

Anticodon loop

Anticodon loop

Anticodon loop

Anticodon end

Figure 15.14 The structure of tRNA. Base-pairing within the molecule creates three stem and loop structures in a characteristic cloverleaf shape. The loop at the bottom of the cloverleaf contains the anticodon sequence, which can base-pair with codons in the mRNA. Amino acids are attached to the free, single-stranded –OH end of the acceptor stem. In its final 3-D structure, the loops of tRNA are folded into the final L-shaped structure. (c): petarg/Shutterstock 304 part III Genetic and Molecular Biology

Amino group NH3+ ATP

Pi Pi

Carboxyl group Trp

C

Charged tRNA travels to ribosome NH

3

O

O− Amino acid site

+

Accepting site

Trp

NH

3

C

AM

O

Trp

AM

P

P O

OH

C

O OH

tRNA

tRNA site

+

NH

3

O AMP

+

NH3+ Trp

C

Trp

C

O

O

O

O

Charged tRNA dissociates

Aminoacyl-tRNA Anticodon synthetase specific to tryptophan 1. In the first step of the reaction, the amino acid is activated. The amino acid reacts with ATP to produce an intermediate with the carboxyl end of the amino acid attached to AMP. The two terminal phosphates (pyrophosphates) are cleaved from ATP in this reaction.

2. The amino acid-AMP complex remains bound to the enzyme. The tRNA next binds to the enzyme.

3. The second step of the reaction transfers the amino acid from AMP to the tRNA, producing a charged tRNA and AMP. The charged tRNA consists of a specific amino acid attached to the 3′ acceptor stem of its RNA.

Figure 15.15 tRNA charging reaction. There are 20 different aminoacyl-tRNA synthetase enzymes each specific for one amino acid,

such as tryptophan (Trp). The enzyme must also recognize and bind to the tRNA molecules with anticodons specifying that amino acid, ACC for tryptophan. The reaction uses ATP and produces an activated intermediate that will not require further energy for peptide bond formation.

90°

Large subunit

3′ E

Small subunit

0°

P

A

Large subunit Small subunit

Large subunit

the ribosome carried out its function and that the rRNA was a structural scaffold necessary to hold the proteins in the correct position. Now this view has mostly been reversed; the ribosome is seen instead as rRNAs that are held in place by proteins. The faces of the two subunits that interact with each other are lined with rRNA, and the parts of both subunits that interact with mRNA, tRNA, and amino acids are also primarily rRNA (figure 15.17). It

mRNA

Small subunit 5′

Figure 15.16 Ribosomes have two subunits. Ribosome

subunits come together and apart as part of a ribosome cycle. The smaller subunit fits into a depression on the surface of the larger one. Ribosomes have three tRNA-binding sites: aminoacyl site (A), peptidyl site (P), and exit site (E).

arranged 5′ to 3′ in the order E, P, and A. The incoming charged tRNAs enter the ribosome at the A site, transit through the P site, and then leave via the E site.

The ribosome has both decoding and enzymatic functions The two functions of the ribosome involve decoding the transcribed message and forming peptide bonds. The decoding function resides primarily in the small subunit of the ribosome. The formation of peptide bonds requires the enzyme peptidyl transferase, which resides in the large subunit. Our view of the ribosome has changed dramatically over time. Initially, molecular biologists assumed that the proteins in

Figure 15.17 3-D structure of eukaryotic ribosome. 

The complete atomic structure of the yeast large ribosomal subunit is shown. The ribosomal RNA is beige, blue and pale green; all other colors are different ribosomal proteins. The faces of each ribosomal subunit are lined with rRNA such that their interaction with tRNAs, amino acids, and mRNA all involve rRNA. Proteins are absent from the active site but abundant everywhere on the surface. The proteins stabilize the structure by interacting with adjacent RNA strands. Science Photo Library/age fotostock

chapter

15 Genes and How They Work 305

is now thought that the peptidyl transferase activity resides in an rRNA in the large subunit.

Learning Outcomes Review 15.6

Initiation requires accessory factors

Transfer RNA has two functional regions, one that bonds with an amino acid, and the other that can base-pair with mRNA. The tRNA charging reaction joins the carboxyl end of an amino acid to the 3′ acceptor stem of its tRNA; without charged tRNAs, translation cannot take place. This reaction is catalyzed by 20 different aminoacyl-tRNA synthetases, one for each amino acid. The ribosome has three different binding sites for tRNA: one for the tRNA adding to the growing peptide chain (P site), one for the next charged tRNA (A site), and one for the previous tRNA, which is now without an amino acid (E site). The ribosome can be thought of as having both a decoding function and an enzymatic function. ■■

As mentioned in section 15.2, the start codon is AUG, which also encodes the amino acid methionine. The ribosome usually uses the first AUG it encounters in an mRNA strand to signal the start of translation.

Prokaryotic initiation In prokaryotes, the initiation complex includes a special i­nitiator tRNA molecule charged with a chemically modified methionine, N-formylmethionine. The initiator tRNA is shown as tRNAfMet. The initiation complex also includes the small ribosomal subunit and the mRNA strand (figure 15.18). The small subunit is positioned correctly on the mRNA due to a conserved sequence in the 5′ end of the mRNA called the ribosome-binding sequence (RBS) that is complementary to the 3′ end of a small subunit rRNA. A number of initiation factors mediate this interaction of the ribosome, mRNA, and tRNAfMet to form the initiation complex. These factors are involved in initiation only and are not part of the ribosome. Once the complex of mRNA, initiator tRNA, and small ribosomal subunit is formed, the large subunit is added, and translation can begin. With the formation of the complete ribosome, the initiator tRNA is bound to the P site with the A site empty.

What would be the effect on translation of a mutant tRNA that had an anticodon complementary to a stop codon?

15.7 The

Process of Translation

Learning Outcomes 1. Describe the process of translation initiation. 2. Explain the elongation cycle. 3. Compare translation on the RER and in the cytoplasm.

Eukaryotic initiation

The process of translation is one of the most complex and energy-expensive tasks that cells perform. An overview of the process is perhaps deceptively simple: the mRNA is threaded through the ribosome, while tRNAs carrying amino acids bind to the ribosome, where they interact with mRNA by base-pairing with the

fMet

3′

mRNA 5′ Small subunit

Initiation in eukaryotes is similar, although it differs in two important ways. First, in eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine. Second, the initiation complex is far more complicated than in prokaryotes, containing nine or more protein factors, many consisting of several subunits. Eukaryotic mRNAs also lack an RBS. The small subunit binds to the mRNA initially by binding to the 5′ cap of the mRNA.

Initiation factor

tRNA in P site

U A C A U G

Large subunit

AUG U A C

mRNA’s codons. The ribosome and tRNAs position the amino acids such that peptide bonds can be formed between each new amino acid and the growing polypeptide.

E site

A site

3′ 3′

3′

Initiation factor 5′

GTP

GDP +

Pi

5′ Initiation complex

5′ Complete ribosome

Figure 15.18 Initiation of translation. In prokaryotes, initiation factors play key roles in positioning the small ribosomal subunit, the

initiator tRNA fMet, and the mRNA. When the tRNA fMet is positioned over the first AUG codon of the mRNA, the large ribosomal subunit binds, forming the E, P, and A sites where successive tRNA molecules bind to the ribosomes, and polypeptide synthesis begins. Ribosomal subunits are shown as a cutaway sectioned through the middle. 306 part III Genetic and Molecular Biology

Elongation adds successive amino acids When the entire ribosome is assembled around the initiator tRNA and mRNA, the second charged tRNA can be brought to the ribosome and bind to the empty A site. This requires an elongation factor called EF-Tu, which binds to the charged tRNA and to GTP. A peptide bond can then form between the amino acid of the initiator tRNA and the newly arrived charged tRNA in the A site. The geometry of this bond relative to the two charged tRNAs is critical to understanding the process. Remember that an amino acid is attached to a tRNA by its carboxyl terminus. The peptide bond is formed between the amino end of the incoming amino acid (in the A site) and the carboxyl end of the growing chain (in the P site) (figure 15.19). The addition of successive amino acids is a series of events that occur in a cyclic fashion. Figure 15.20 shows the details of the elongation cycle: 1. Matching tRNA anticodon with mRNA codon. Each new charged tRNA comes to the ribosome bound to EF-Tu and GTP. The charged tRNA binds to the A site if its anticodon is complementary to the mRNA codon in the A site. After binding, GTP is hydrolyzed, and EF-Tu–GDP dissociates from the ribosome, to be recycled by another factor. This two-step binding and hydrolysis of GTP is thought to increase the accuracy of translation. 2. Peptide bond formation. Peptidyl transferase, located in the large subunit, catalyzes the formation of a peptide

bond between the amino group of the amino acid in the A site and the carboxyl group of the growing chain. This also breaks the bond between the growing chain and the tRNA in the P site, leaving it empty (no longer charged). The overall result of this is to transfer the growing chain to the tRNA in the A site. 3. Translocation of the ribosome. After the peptide bond has been formed, the ribosome moves relative to the mRNA and the tRNAs. The next codon in the mRNA shifts into the A site, and the tRNA with the growing chain moves to the P site. The uncharged tRNA f­ ormerly in the P site is now in the E site, and it will be ejected in the next cycle. This translocation step requires the accessory factor EF-G and the hydrolysis of another GTP. This elongation cycle continues with each new amino acid added. The ribosome moves down the mRNA in a 5′-to-3′ direction, reading successive codons. The tRNAs move through the ribosome in the opposite direction, from the A site to the P site and finally the E site, before they are ejected as empty tRNAs, which can be charged with another amino acid and then used again.

Wobble pairing As mentioned, there are fewer tRNAs than codons. This situation is easily rationalized because the pairing between the 3′ base of the codon and the 5′ base of the anticodon is less stringent than normal. In some tRNAs, the presence of modified

NH3+ Amino group Amino acid 1 O NH3

+

O

C

C

O

O

Peptide bond formation

“Empty” tRNA

OH

Amino acid 1

Peptide bond

N

Amino acid 2

Amino acid 2 C

O

NH3+

C

Amino acid 2

Amino acid 1 3′

Polypeptide chain

NH3+

Amino end (N terminus)

Amino acid 3

O

Amino acid 4

O

Amino acid 5 Amino acid 6 Amino acid 7

5′ COO− P site

A site

P site

A site

Carboxyl end (C terminus)

Figure 15.19 Peptide bond formation. Peptide bonds are formed between a “new” charged tRNA in the A site and the growing chain attached to the tRNA in the P site. The bond forms between the amino group of the new amino acid and the carboxyl group of the growing chain. This breaks the bond between the growing chain and its tRNA, transferring it to the A site as the new amino acid remains attached to its tRNA. chapter

15 Genes and How They Work 307

Figure 15.20 Elongation cycle. 

3′

3′

d

g tRNA antico do nw ith

Numbering of the cycle corresponds to the numbering in the text. The cycle begins when a new charged tRNA with anticodon matching the codon of the mRNA in the A site + Pi GDP arrives with EF-Tu. The EF-Tu Elongation hydrolyzes GTP and dissociates factor A P from the ribosome. A peptide E bond is formed between the Elongation amino acid in the A site and 5′ factor GTP the growing chain in the P 2. Pe on ptid cod site, transferring the A eb 3′ RN on m growing chain to the A site, and leaving the tRNA in the P site empty. Ribosome A P E translocation requires 5′ another elongation factor Sectioned ribosome and GTP hydrolysis. This moves the tRNA in GTP the A site into the P site, Next round Elongation the next codon in the factor o mRNA into the A site, ati oc l “Ejected” tRNA s and the empty tRNA into an 3. Tr the E site.

the ribo some

n

of

Ma 1.

n tchi

n atio rm fo

E 5′

GTP

P

3′

A E

5′

Elongation factor GDP + Pi

Growing polypeptide

3′

E

A

P

P

A

5′

bases with less accurate pairing in the 5′ position of the anticodon enhances this flexibility. This effect is referred to as wobble pairing because these tRNAs can “wobble” a bit on the mRNA, so that a single tRNA can “read” more than one codon in the mRNA.

?

Inquiry question How is the wobble phenomenon

related to the number of tRNAs and the degeneracy of the genetic code?

Termination requires accessory factors Elongation continues in this fashion until a chain-terminating stop codon is reached (for example, UAA in figure 15.21). These stop codons do not bind to tRNA; instead, they are recognized by release factors, proteins that release the newly made polypeptide from the ribosome.

Proteins may be targeted to the ER In eukaryotes, translation can occur either in the cytoplasm or on the RER. Proteins that are translated on the RER are targeted there 308 part III Genetic and Molecular Biology

based on their own initial amino acid sequence. The ribosomes found on the RER are actively translating and are not permanently bound to the ER. A polypeptide that starts with a short series of amino acids called a signal sequence is specifically recognized and bound by a cytoplasmic complex of proteins called the signal recognition particle (SRP). The complex of signal sequence and SRP is in turn recognized by a receptor protein in the ER membrane. The binding of the ER receptor to the signal sequence/SRP complex holds the ribosome engaged in translation of the protein on the ER membrane, a process called docking (figure 15.22). As the protein is assembled, it passes through a channel formed by the docking complex and into the interior ER compartment, the cisternal space. This is the basis for the docking metaphor—the ribosome is not actually bound to the ER itself, but with the newly synthesized protein entering the ER, the ribosome is like a boat tied to a dock with a rope. The basic mechanism of protein translocation across membranes by the SRP and its receptor and channel complex has been conserved across all three cell types: eukaryotes, bacteria, and archaea. Given that only eukaryotic cells have an

endomembrane system, this universality may seem curious; however, bacteria and archaea both export proteins through their plasma membrane, and the mechanism used is similar to the way in which eukaryotes move proteins into the cisternal space of the ER. Once within the ER cisternal space, or lumen, the newly synthesized protein can be modified by the addition of sugars (glycosylation) and transported by vesicles to the Golgi apparatus (see chapter 4). This is the beginning of the protein-trafficking pathway that can lead to other intracellular targets, to incorporation into the plasma membrane, or to release outside of the cell itself.

Polypeptide chain releases Dissociation

3′

Release factor

5′ 3′

Learning Outcomes Review 15.7

Translation initiation involves the interaction of the small ribosomal subunit with mRNA and a charged initiator tRNA. The elongation cycle involves bringing in new charged tRNAs to the ribosome’s A site, forming peptide bonds between amino acids, and translocating the ribosome along the mRNA chain. The tRNAs transit through the ribosome from A to P to E sites during the process. In eukaryotes, signal sequences of a newly forming polypeptide may target it and its ribosome to be moved to the RER. Polypeptides formed on the RER enter the cisternal space rather than being released into the cytoplasm.

5′ Sectioned ribosome

C A C G G U E

P

A U A A

■■

Figure 15.21 Termination of protein synthesis. There is

What stages of translation require energy?

no tRNA with an anticodon complementary to any of the three termination signal codons. When a ribosome encounters a termination codon, it stops translocating. A specific protein release factor facilitates the release of the polypeptide chain by breaking the covalent bond that links the polypeptide to the P site tRNA. Rough endoplasmic reticulum (RER)

Figure 15.22 Synthesis of proteins on RER.  Proteins that are synthesized on RER arrive at the ER because of sequences in the peptide itself. A signal sequence in the amino terminus of the polypeptide is recognized by the signal recognition particle (SRP). This complex docks with a receptor associated with a channel in the ER. The peptide passes through the channel into the lumen of the ER as it is synthesized.

SRP binds to signal peptide, arresting elongation

Cytoplasm

Lumen of the RER

Protein channel

Docking

NH2 Polypeptide elongation continues

Signal recognition particle (SRP) Signal Exit tunnel

Ribosome synthesizing peptide chapter

15 Genes and How They Work 309

15.8 Summarizing

Expression

■■

Gene

■■

Learning Outcome 1. Explain the entire process of gene expression.

Because of the complexity of the process of gene expression, it is worth stepping back to summarize some key points: ■■

■■

The process of gene expression converts information in the genotype into the phenotype. A copy of the gene in the form of mRNA is produced by transcription, and the mRNA is used to direct the synthesis of a protein by translation. 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript.

Both transcription and translation can be broken down into initiation, an elongation cycle, and termination—processes that produce their respective polymers. (The same is true for DNA replication.) Eukaryotic gene expression is much more complex than that of prokaryotes.

The structure of eukaryotic genes with interrupted coding sequences complicates both the process of gene expression and the nature of genetic information. It means that processing must occur between transcription and translation, and that one gene can produce multiple messages. Transcription in eukaryotes also takes place in the nucleus, whereas translation takes place in the cytoplasm. This necessitates that the mRNA be transported through nuclear pores to the cytoplasm prior to translation. The entire eukaryotic process is summarized in figure 15.23, and differences in gene expression between prokaryotes and in eukaryotes are summarized in table 15.2.

Figure 15.23 An overview of gene expression in eukaryotes.

RNA polymerase II

3′

5′

2. The primary transcript is processed by addition of a 5′ methyl-G cap, cleavage and polyadenylation of the 3′ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm.

Primary RNA transcript

Primary RNA transcript Poly-A tail

Cut intron

3. The 5′ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex.

Amino acids

tRNA arrives in A site 3′ mRNA

5′

A site P site E site

4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site.

310 part III Genetic and Molecular Biology

Cytoplasm

Large subunit 5′ cap

mRNA

Small subunit Cytoplasm

Empty tRNA moves into E site and is ejected

Lengthening polypeptide chain

Empty tRNA

Mature mRNA 5′ cap

3′

3′

5′ 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty.

5′ 6. Ribosome translocation moves the ribosome relative to the mRNA and its bound tRNAs. This moves the growing chain into the P site, leaving the empty tRNA in the E site and the A site ready to bind the next charged tRNA.

TA B L E 1 5 . 2

Differences Between Prokaryotic and Eukaryotic Gene Expression

Characteristic

Prokaryotes

Eukaryotes

Introns

No introns, although some archaeal genes possess them.

Most genes contain introns.

Number of genes in mRNA

Several genes may be transcribed into a single mRNA molecule. Often these have related functions and form an operon, which helps coordinate regulation of biochemical pathways.

Only one gene per mRNA molecule; regulation of pathways accomplished in other ways.

Site of transcription and translation

No membrane-bounded nucleus; transcription and translation are coupled.

Transcription in nucleus; mRNA is transported to the cytoplasm for translation.

Initiation of translation

Begins at AUG codon preceded by special sequence that binds the ribosome.

Begins at AUG codon preceded by the 5′ cap (methylated GTP) that binds the ribosome.

Modification of mRNA after transcription

None; translation begins before transcription is completed. Transcription and translation are coupled.

A number of modifications while the mRNA is in the nucleus: introns are removed and exons are spliced together; a 5′ cap is added; a poly-A tail is added.

The term mutation is defined as a heritable change to the genetic material. Traditionally this was detected by a change in phenotype, but now in the age of genome sequencing, mutations can be detected in DNA. Changing a single base in a gene can have profound consequences. For example, in sickle cell anemia (see chapter 13), a change of A to T in the β-globin gene results in a glutamic acid being replaced by valine. Because valine is nonpolar, this causes the β chains to aggregate into polymers, altering the shape of the cells, ultimately producing the clinical syndrome (figure 15.24). Genetic differences between individuals in a population are called polymorphisms, which arise by the process of mutation. We saw in chapter 13 that single-nucleotide polymorphisms are useful in mapping genes. In this section, we will consider the range of changes that can occur in the genome; then in chapter 18 we will look at the range of variation this has produced in human populations.

Learning Outcomes Review 15.8

The greater complexity of eukaryotic gene expression is related to the functional organization of the cell, with DNA in the nucleus and ribosomes in the cytoplasm. The differences in gene expression between prokaryotes and eukaryotes is mainly in detail, but some differences have functional significance.

15.9 Mutation:

Altered Genes

Learning Outcomes

Point mutations produce single-nucleotide variation (SNV)

1. Describe the effects of different point mutations. 2. Explain how chromosomal alterations can change copy number. 3. Describe the different kinds of structural variation.

A mutation that alters a single base is termed a point mutation, which leads to single-nucleotide variation (SNV) in populations; Normal Deoxygenated Tetramer

Normal HBB Sequence Polar Leu

C

T

Thr

G

A

C

Pro

T

C

C

Glu

T

G

A

Glu

G

G

A

Lys

G

A

A

Ser

G

T

C

Amino acids

T Nucleotides

Abnormal Deoxygenated Tetramer

α1

α2

α1

α2

β1

β2

β1

β2

Hemoglobin tetramer

“Sticky” nonpolar sites

Abnormal HBB Sequence Nonpolar (hydrophobic) Leu

C

T

Thr

G

A

C

Pro

T

C

C

Val

T

G

T

Glu

G

G

A

Lys

G

A

A

Ser

G

T

C

Amino acids

T Nucleotides

Tetramers form long chains when deoxygenated. This distorts the normal red blood cell shape into a sickle shape.

Figure 15.24 Sickle cell anemia is caused by an altered protein. Hemoglobin is composed of a tetramer of two α-globin and two β-globin chains. The sickle cell allele of the β-globin gene contains a single base change resulting in the substitution of Val for Glu. This creates a hydrophobic region on the surface of the protein that is “sticky,” leading to their association into long chains that distort the shape of the red blood cells. chapter

15 Genes and How They Work 311

as these are polymorphisms in populations, they are also called single-nucleotide polymorphisms (SNP). The replacement of one base for another is called a base substitution mutation (figure 15.25). Base substitutions fall into two categories based on whether the new base is similar to the old one. If the change replaces a purine with another purine (for example, A to G), or a pyrimidine with another pyrimidine (for example, C to T), we call this a transition. If the change replaces a purine with a pyrimidine or the reverse (for example, G to C), we call this a transversion.

Missense mutations Because the genetic code is degenerate, a base substitution mutation may not change the encoded amino acid. If it does not, we say the mutation is synonymous, or silent (figure 15.25b). If it changes an amino acid, we say the mutation is nonsynonymous. We also refer to nonsynonymous changes as missense mutations, as the “sense” of the codon involved has been changed (figure 15.25c). We further classify nonsynonymous changes as conservative or nonconservative. Conservative changes replace an amino acid with a chemically similar one (nonpolar for nonpolar, for example) while nonconservative changes replace an amino acid with a chemically different one (charged for uncharged, for example).

C G Coding Template

3′–TACGGAATAGCGACT–5′ 5′–AUGCCUUAUCGC UGA–3′

Protein

312 part III Genetic and Molecular Biology

Met Pro Tyr Arg Stop

a. Silent Mutation C G Coding

5′–ATGCCCTATCGCTGA–3′

Template

3′–TACGGGATAGCGACT–5′

mRNA

5′–AUGCCCUAUCGCUGA–3′

Protein

Met Pro Tyr Arg Stop

b. Missense Mutation

A mutation that changes a codon from one that encodes an amino acid to a stop codon is called a nonsense mutation because the codon no longer makes “sense” to the translation apparatus (figure  15.25d). The stop codon will cause translation to terminate prematurely, producing a truncated protein. The length of the resulting protein depends on the location of the new stop codon in the gene.

The gain or loss of 1–50 bp is called an indel (for insertion/deletion). Since codons consist of three bases, insertions or deletions that do not occur in multiples of three will shift the reading frame in the mRNA. Such a frameshift mutation randomizes the downstream sequence of amino acids. The consequences for protein function depend on the location of the frameshift, but they usually inactivate the protein. Frameshift mutations often lead to premature termination as 3 in 64 codons are stop codons, or roughly a stop every 20 amino acids in a random sequence. In section 15.2, we saw how Crick and Brenner used frameshift mutations to infer the triplet nature of the genetic code. A special case of insertions is a class of mutation first discovered in Huntington disease. The gene involved contains a sequence of three bases that are repeated, called a trinucleotide repeat (TNR) or triplet repeat. This repeat sequence is expanded in the disease allele relative to the normal allele. This kind of triplet repeat expansion has been found in other neurodegenerative disorders, and repeat expansion disorders share some unusual features of inheritance. The prevalence of this kind of mutation is unclear, but at present they have only been observed in humans and mice, implying that they may be limited to vertebrates, or even mammals.

5′–ATGCCT TAT CGCTGA–3′

mRNA

Nonsense mutations

Small insertions or deletions are called indels

A A T T

A T Coding

5′–ATGCCTTATC ACTGA–3′

Template

3′–TACGGAATAGTGACT–5′

mRNA

5′–AUGCCUUAUCACUGA–3′

Protein

Met Pro Tyr His Stop

c. Nonsense Mutation A T Coding

5′–ATGCCT TAACGCTGA–3′

Template

3′–TACGGAAT TGCGACT–5′

mRNA

5′–AUGCCUUA A CGC UGA–3′

Protein

Met Pro Stop

d.

Figure 15.25 Types of point mutations. a. A hypothetical

gene is shown with encoded mRNA and protein. Arrows above the gene indicate sites of mutations described in the rest of the figure. b.  Silent mutation. Due to degeneracy in the genetic code, base substitution does not always change an amino acid. This usually involves the third position of a codon, in this case a T/A to C/G mutation. c. Missense mutation. The G/C to A/T mutation changes the amino acid encoded from arginine to histidine. d. Nonsense mutation. The T/A to A/T mutation produces a UAA stop codon in the mRNA.

Triplet repeat expansion can occur in the coding region or in noncoding transcribed DNA. In the case of Huntington disease, the repeat unit is in the coding region of the gene, and the triplet encodes glutamine, and expansion results in a polyglutamine region in the protein. A number of other neurodegenerative disorders are due to this kind of mutation.

Deletion Deleted A B C D E F G H I J

a.

Larger changes lead to structural variation (SV) Point mutations affect a single site in a chromosome, but more extensive changes that affect more than 50 bp are called structural variation. Many human cancers are associated with chromosomal abnormalities, so these can be clinically relevant. We will consider the nature of structural variation here, and then consider the extent of this variation in humans in chapter 18.

Duplication Duplicated A B C D E F G H I J

Balanced rearrangements do not change copy number Material in a chromosome can become inverted, or transferred between chromosomes, events call inversions and reciprocal translocations respectively (figure 15.26 c,d). In each case, the total amount of genetic material remains the same, but it has been rearranged. More complex rearrangements can also occur, but they are even more rare. An inversion results when a segment of a chromosome is broken in two places, reversed, and put back together. An inversion may not have an effect on phenotype if the sites where the inversion occurs do not break within a gene. In fact, although humans all have the “same” genome, the order of genes in all individuals in a population is not precisely the same due to inversions that occur in different lineages.

A B C D B C D E F G H I J

b. Inversion

Copy number variation (CNV) When genomic rearrangements result in differences in the number of copies of a particular genomic region, it is called copy number variation (CNV). This can be the result of loss of DNA by deletion, or the gain of DNA by duplication (figure 15.26 a,b) or insertion. A special case of possible copy number variation is the insertion of mobile genetic elements, or mobile element insertion (MEI). The amount of DNA that can be deleted without drastic consequences is dependent on the region of the genome, but large deletions are usually lethal. One human syndrome caused by a deletion is cri-du-chat, which is French for “cry of the cat” after the noise made by children with this syndrome. Cri-du-chat syndrome is caused by a large deletion from the short arm of chromosome 5. It usually results in early death, although many affected individuals show a normal life span. It has a variety of effects, including respiratory problems. The duplication of a chromosomal region may or may not lead to phenotypic consequences. Effects depend upon the location of the “breakpoints” where the duplication occurred. If the duplicated region does not lie within a gene, there may be no effect. If the duplication occurs next to the original region, it is termed a tandem duplication. Some gene families, such as the globin genes that encode hemoglobin, arose as the result of tandem duplication.

A E F G H I J

Inverted A B C D E F G H I J

A D C B E F G H I J

c. Reciprocal Translocation A B C D E F G H I J

K L M D E F G H I J

K L M N OP Q R

A B C N OP Q R

d.

Figure 15.26 Chromosomal mutations. Larger-scale changes in chromosomes are also possible. Material can be deleted (a), duplicated (b), and inverted (c). Translocations occur when one chromosome is broken and becomes part of another chromosome. This often occurs where both chromosomes are broken and exchange material, an event called a reciprocal translocation (d). If a piece of one chromosome is broken off and joined to another chromosome, we call this a translocation. Translocations are complex because they cause problems during meiosis, when two different chromosomes try to pair with each other during meiosis, which can lead to unbalanced translocation chromosomes. Translocations can also move genes from one chromosomal region to another in a manner that changes the expression of genes in the region involved. Two forms of leukemia have been shown to be associated with translocations that move oncogenes into regions of a chromosome where they are expressed inappropriately in blood cells.

Mutations are the starting point of evolution Without a continuous source of new variation, evolution would not occur. However, mutations are much more often detrimental than beneficial to an individual. Thus a delicate balance must exist between the amount of new variation that arises in a species, and the

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15 Genes and How They Work 313

health of individuals in the species. We will explore these ideas in more detail in chapter 20. The larger-scale alteration of chromosomes has also been important in evolution, although its role is poorly understood. It is clear that gene families arise by the duplication of an ancestral gene, followed by the functional divergence of the duplicated copies. It is also clear that even among closely related species, the number and arrangements of genes on chromosomes can differ. There have even been whole genome duplications in lineages.

Human mutation rates have been directly measured The mutation rate in humans was first estimated by J. B. S. Haldane, who used the prevalence of hemophilia in the population. Whole-genome sequencing now allows direct measurement of mutation rate by sequencing large numbers of parent–offspring trios (trio = mom, dad, child). Multiple studies of this sort estimate a mutation rate of 1.0−1.8 × 10−8 per nucleotide per generation. To put this in simpler terms, this represents 44–82 new SNV mutations in the average genome, with one to two of these affecting coding sequences. These data also show a strong paternal bias: about 80% of new germline point mutations arise in the paternal genome. This rate also increases with increasing paternal age. Much like the prevalence of aneuploidy in the maternal germ line, this is probably a

reflection of reproductive biology: the stem cell population that gives rise to sperm accumulates mutations with age. Indels occur at a rate about one-fifth to one-tenth the SNV rate. Much larger deletions or duplications, duplications that cause CNV, occur at an even lower rate. This is estimated at about 1 new large CNV per 42 births. Some of the mobile genetic elements that litter our genome (see chapter 18) can still move. The rate for mobile element insertion appears to be on the order of about 1 per 20 births. These data will continue to shed light on human evolution, especially combined with analysis of other primate species. It is also relevant to human genetic diseases, as it now appears that de novo mutations may play a larger role than previously thought.

Learning Outcomes Review 15.9

Point mutations, or single-nucleotide variation, include missense mutations that cause substitution of one amino acid for another, and nonsense mutations that halt transcription. Indels are insertions or deletions of up to 50 bp. This may cause a change in reading frame, or frameshift, with drastic consequences. Structural variation can change copy number or be balanced. Deletions and duplications change copy number, while inversions and reciprocal translocations alter chromosomes but do not change copy number. ■■

Would an inversion or duplication always be expected to have a phenotype?

Chapter Review Dr. Gopal Murti/Science Source

15.1 The Nature of Genes

15.2 The Genetic Code

Garrod concluded that inherited disorders can involve specific enzymes. Garrod found that alkaptonuria is due to an altered enzyme.

The code is read in groups of three. Crick and Brenner showed that the code is nonoverlapping and is read in groups of three. This finding established the concept of reading frame.

Beadle and Tatum showed that genes specify enzymes. Neurospora mutants unable to synthesize arginine were found to lack specific enzymes. Beadle and Tatum advanced the “one gene/one polypeptide” hypothesis (figure 15.1). The central dogma describes information flow in cells as DNA to RNA to protein (figure 15.2). We call the DNA strand copied to mRNA the template (antisense) strand; the other, the coding (sense) strand. Transcription makes an RNA copy of DNA. Translation uses information in RNA to synthesize proteins. An adapter molecule, tRNA, is required to connect the information in mRNA into the sequence of amino acids. RNA has multiple roles in gene expression. 314 part III Genetic and Molecular Biology

Nirenberg and others deciphered the code. A codon consists of 3 nucleotides, so there are 64 possible codons. Three codons signal “stop,” and one codon signals “start” and also encodes methionine. Thus 61 codons encode the 20 amino acids. The code is degenerate but specific. Many amino acids have more than one codon, but each codon specifies only a single amino acid. The code is practically universal, but not quite. In some mitochondrial and protist genomes, a stop codon is read as an amino acid; otherwise the code is universal.

15.3 Prokaryotic Transcription

15.6 The Structure of tRNA and Ribosomes

Prokaryotes have a single RNA polymerase. Prokaryotic RNA polymerase exists in two forms: core polymerase, which can synthesize mRNA; and holoenzyme, core plus σ factor, which can accurately initiate synthesis (figure 15.6).

Aminoacyl-tRNA synthetases attach amino acids to tRNA. The tRNA charging reaction attaches the carboxyl terminus of an amino acid to the 3′ end of the correct tRNA (figure 15.15).

Initiation occurs at promoters. Initiation requires a start site and a promoter. The promoter is upstream of the start site, and binding of RNA polymerase holoenzyme to its –35 region positions the polymerase properly. Elongation adds successive nucleotides. Transcription proceeds in the 5′-to-3′ direction. The transcription bubble contains RNA polymerase, the locally unwound DNA template, and the growing mRNA transcript (figure 15.7). Termination occurs at specific sites. Terminators consist of complementary sequences that form a doublestranded hairpin loop where the polymerase pauses (figure 15.8). Prokaryotic transcription is coupled to translation. Translation begins while mRNAs are still being transcribed.

The ribosome has multiple tRNA-binding sites (figure 15.16). A charged tRNA first binds to the A site, then moves to the P site where its amino acid is bonded to the peptide chain, and finally, without its amino acid, moves to the E site from which it is released. The ribosome has both decoding and enzymatic functions. Ribosomes hold tRNAs and mRNA in position for a ribosomal enzyme to form peptide bonds.

15.7 The Process of Translation Initiation requires accessory factors. In prokaryotes, initiation-complex formation is aided by the ribosomebinding sequence (RBS) of mRNA, complementary to a small subunit. Eukaryotes use the 5′ cap for the same function.

15.4 Eukaryotic Transcription

Elongation adds successive amino acids (figure 15.20). As the ribosome moves along the mRNA, new amino acids from charged tRNAs are added to the growing peptide.

RNA polymerase I transcribes rRNA; polymerase II transcribes mRNA and some snRNAs; polymerase III transcribes tRNA.

Termination requires accessory factors. Stop codons are recognized by termination factors.

Eukaryotes have three RNA polymerases.

Proteins may be targeted to the ER. In eukaryotes, proteins with a signal sequence in their amino terminus bind to the SRP, and this complex docks on the ER.

The core promoter binds factors that recruit RNA Pol II. General transcription factors bind to the promoter and recruit the polymerase to form the initiation complex. As the transcript reaches about 20 nucleotides, it is capped, and the polymerase may pause. Release of pausing requires elongation factors. The transcription elongation complex can recruit other factors. Some general factors in the initiation complex are replaced by elongation factors. The carboxy terminal domain (CTD) interacts with elongation factors and enzymes that modify the transcript. Polyadenylation is involved in terminating mRNA transcripts. The enzyme poly-A polymerase cleaves the transcript and adds 1 to 200 adenine (A) residues to the 3′ end. Primary transcripts are modified to produce mRNAs. The mRNA has a 5′ cap and a 3′ poly-A tail, and introns are removed.

15.5 Eukaryotic pre-mRNA Splicing Eukaryotic genes may contain interruptions. Coding DNA (an exon) is interrupted by noncoding introns. These introns are removed by splicing (figure 15.13). The spliceosome is a splicing machine. snRNPs recognize intron–exon junctions and recruit spliceosomes. The spliceosome ultimately joins the 3′ end of the first exon to the 5′ end of the next exon.

15.8 Summarizing Gene Expression 15.9 Mutation: Altered Genes Point mutations produce single-nucleotide variation (SNV). Base substitutions exchange one base for another, which may not change encoded amino acids. Missense mutations change amino acids, while nonsense mutations create stop codons. Small insertions or deletions are called indels. Gain or loss of 1-50 bp is called an indel. If not a multiple of 3, they will cause frameshifts, which often lead to shortened proteins. Triplet repeat expansion is a special case of insertion found in neurodegenerative disorders. Larger changes lead to structural variation (SV). Chromosomal mutations include additions, deletions, inversions, and translocations. Mutations are the starting point of evolution. Human mutation rates have been directly measured. There are about 70 new mutations per generation.

Splicing can produce multiple transcripts from the same gene.

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15 Genes and How They Work 315

Visual Summary Gene expression

Central dogma DNA→RNA→Protein

RNA processing

Transcription requires

uses

Eukaryotes

Ribosome

steps Universal Initiation

consists of

64 codons

code for

Amino acids

Start

also includes

5′ cap

Pre-mRNA splicing

Promoter

and

Transcription factors

Degenerate Specific

includes

uses

Triplet

3′ poly-A tail Elongation

requires

occurs in

RNA polymerase

Genetic code

Translation

5′→3′

steps Anticodon ccan result in

requires

Initiation

Alternative splicing

Spliceosome

contains Charged tRNA

Elongation snRNPs

Stop

Termination

Termination

AUG

removes

Introns

joins

Exons

Peptidyl transferase

Review Questions Dr. Gopal Murti/Science Source

U N D E R S TA N D 1. The experiments with nutritional mutants in Neurospora by Beadle and Tatum provided evidence that a. b. c. d.

bread mold can be grown in a lab on minimal media. X-rays can damage DNA. cells need enzymes. genes specify enzymes.

2. What is the central dogma of molecular biology? a. b. c. d.

DNA is the genetic material. Information passes from DNA directly to protein. Information passes from DNA to RNA to protein. One gene encodes only one polypeptide.

3. In the genetic code, one codon a. b. c. d.

consists of three bases. specifies a single amino acid. specifies more than one amino acid. Both a and b are correct.

316 part III Genetic and Molecular Biology

4. Eukaryotic transcription differs from prokaryotic in that a. b. c. d.

eukaryotes have only one RNA polymerase. eukaryotes have three RNA polymerases. prokaryotes have three RNA polymerases. Both a and c are correct.

5. An anticodon would be found on which of the following types of RNA? a. b.

snRNA (small nuclear RNA) mRNA (messenger RNA)

c.  tRNA (transfer RNA) d.  rRNA (ribosomal RNA)

6. RNA polymerase binds to a _________ to initiate _________. a. b. c. d.

mRNA; translation promoter; transcription primer; transcription transcription factor; translation

7. During translation, the codon in mRNA is actually “read” by a. b.

the A site in the ribosome. c.  the anticodon in a tRNA. the P site in the ribosome. d.  the anticodon in an amino acid.

A P P LY 1. You have mutants that all affect the same biochemical pathway. If feeding an intermediate in the pathway supports growth, this tells you that the enzyme encoded by the affected gene a. b. c. d.

acts after the intermediate used. acts before the intermediate used. must act to produce the intermediate. must not act to produce the intermediate.

2. The splicing process a. b. c. d.

occurs in prokaryotes. joins introns together. can produce multiple mRNAs from the same transcript. only joins exons for each gene in one way.

3. The enzyme that forms peptide bonds is called peptidyl transferase because it transfers a. b. c. d.

a new amino acid from a tRNA to the growing peptide. the growing peptide from a tRNA to the next amino acid. the peptide from one amino acid to another. the peptide from the ribosome to a charged tRNA.

4. In comparing gene expression in prokaryotes and eukaryotes a. b. c. d.

eukaryotic genes can produce more than one protein. prokaryotic genes can produce more than one protein. both produce mRNAs that are colinear with the protein. Both a and c are correct.

5. The codon CCA could be mutated to produce a. b. c. d.

a silent mutation. a codon for Lys. a stop codon. Both a and b are correct.

7. What is the relationship between mutations and evolution? a. b. c. d.

Mutations make genes better. Mutations can create new alleles. Mutations happened early in evolution, but not now. There is no relationship between evolution and genetic mutations.

SYNTHESIZE 1. A template strand of DNA has the following sequence: 3′ – CGTTACCCGAGCCGTACGATTAGG – 5′ Use the sequence information to determine a. the predicted sequence of the mRNA for this gene. b. the predicted amino acid sequence of the protein. 2. Frameshift mutations often result in truncated proteins. Explain this observation based on the genetic code. 3. Describe how each of the following mutations will affect the final protein product (protein begins with start codon). Name the type of mutation. Original template strand: 3′ – CGTTACCCGAGCCGTACGATTAGG – 5′ a. 3′ – CGTTACCCGAGCCGTAACGATTAGG – 5′ b. 3′ – CGTTACCCGATCCGTACGATTAGG – 5′ c. 3′ – CGTTACCCGAGCCGTTCGATTAGG – 5′ 4. There are a number of features that are unique to bacteria, and others that are unique to eukaryotes. Could any of these features offer the possibility to control gene expression in a way that is unique to either eukaryotes or bacteria?

6. An inversion will a. b. c. d.

necessarily cause a mutant phenotype. only cause a mutant phenotype if the inversion breakpoints fall within a gene. halt transcription in the inverted region because the chromosome is now backward. interfere with translation of genes in the inverted region.

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15 Genes and How They Work 317

16 CHAPTER

Control of Gene Expression Chapter Contents 16.1

Control of Gene Expression

16.2

Regulatory Proteins

16.3

Prokaryotic Regulation

16.4

Eukaryotic Regulation

16.5

Chromatin Structure Affects Gene Expression

16.6

Eukaryotic Posttranscriptional Regulation

16.7

Protein Degradation

6 μm

Science Source

Introduction

Visual Outline requires Regulatory proteins

respond to

Control of gene expression

Prokaryotic cell

in

Environmental changes

Eukaryotic cell involves

examples Initiation complex

include

Repressor

lac operon

trp operon

Activator

lac repressor

trp repressor

binds

binds

CAP activator

Operator

Chromatin structure

Transcription factors

General

Specific

Posttranslational regulation

B

TAFs F TFIID

A

H

E

In a symphony, various instruments play their own parts at different times; the musical score determines which instruments play when. Similarly, in an organism, different genes are expressed at different times, with a “genetic score,” written in regulatory regions of the DNA, determining which genes are active when. The picture shows the expanded “puff” of this Drosophila chromosome, which represents genes that are being actively expressed. Gene expression and how it is controlled is our topic in this chapter.

 16.1

Control of Gene Expression

Learning Outcomes 1. Identify when gene expression is usually controlled. 2. Describe the usual action of regulatory proteins. 3. List differences between control of gene expression in prokaryotes and eukaryotes.

Control of gene expression is essential to all organisms. In prokaryotes, cells adapt to environmental changes by changing gene expression. In multicellular eukaryotes, it is critical for directing development and maintaining homeostasis. In fact, different cell types in a multicellular organism have distinct functions based on the proteins they express. All cells in a multicellular organism share a set of “housekeeping” genes that sustain the cell, but they also express a unique set of proteins that distinguish each cell type. Unicellular eukaryotes also use different control mechanisms from those of prokaryotes. All eukaryotes have a membranebounded nucleus, use similar mechanisms to condense DNA into chromosomes, and have the same gene expression machinery, all of which differ from those of prokaryotes.

Control can occur at all levels of gene expression You learned in chapter 15 that gene expression is the conversion of genotype to phenotype—the flow of information from DNA to produce functional proteins that control cellular activities. This traditional view of gene expression includes controlling the process primarily at the level of transcription initiation. Although this view remains valid, evidence is accumulating that both the extent of the genome transcribed and the control of this transcription in multicellular organisms are even more complex than previously expected. In this chapter, we will develop the control of initiation of transcription in some detail because of its importance, and because it is still the best-studied mechanism for the control of gene expression. With this framework in place, we will also consider how chromatin structure affects gene expression and how control can be exerted posttranscriptionally as well. The latter topic will lead us into the exciting world of regulatory RNA molecules. RNA polymerase is key to transcription, and it must have access to the DNA helix and must be capable of binding to the gene’s promoter for transcription to begin. Regulatory ­proteins act by modulating the ability of RNA polymerase to bind to the promoter. This idea of controlling the access of RNA polymerase to a promoter is common to both prokaryotes and eukaryotes, but the details differ greatly, as you will see. These regulatory proteins bind to specific nucleotide sequences on the DNA that are usually only 10 to 15 nt in length. (Even a large regulatory protein has a “footprint,” or binding area, of only about 20 nt.) Hundreds of these regulatory sequences have been characterized, and each provides a binding site for a specific protein that is able to recognize the sequence. Binding of the protein either blocks transcription by getting in the way of RNA

polymerase or stimulates transcription by facilitating the binding of RNA polymerase to the promoter.

Control strategies in prokaryotes are geared to adjust to environmental changes Control of gene expression is accomplished very differently in prokaryotes than it is in eukaryotes. Prokaryotic cells have been shaped by evolution to grow and divide as rapidly as possible, enabling them to exploit transient resources. Proteins in prokaryotes turn over rapidly, allowing these organisms to respond quickly to changes in their external environment by changing patterns of gene expression. In prokaryotes, the primary function of gene control is to adjust the cell’s activities to its immediate environment. Changes in gene expression alter which enzymes are present in response to the quantity and type of nutrients and the amount of oxygen available. Almost all of these changes are fully reversible, allowing the cell to adjust its enzyme levels up or down in response to environment changes.

Control strategies in eukaryotes maintain homeostasis and drive development The cells of multicellular organisms, in contrast, have been shaped by evolution to be protected from transient changes in their immediate environment. Most of them experience fairly constant conditions. Indeed, homeostasis—the maintenance of a constant internal environment—is a hallmark of multicellular organisms. Cells in such organisms respond to signals in their immediate environment (such as growth factors and hormones) by altering gene expression, and in doing so they participate in regulating the body as a whole. Perhaps the most important role for controlled changes in gene expression is the development of the organism itself. This occurs by coordinated changes in gene expression that occur both over developmental time and in different tissues. Understanding this complex program has been a major goal of developmental genetics, and this work has uncovered circuits of gene expression that have been preserved over very long evolutionary time (see chapter 19). The same regulatory circuit may also be used within an organism to pattern different structures in different places at different developmental times. For example, the gene sonic hedgehog acts to pattern the neural tube, then later to pattern digits in developing limbs.

Learning Outcomes Review 16.1

Gene expression is usually controlled at the level of transcription initiation. Regulatory proteins bind to specific DNA sequences and affect the binding of RNA polymerase to promoters. Individual proteins may either prevent or stimulate transcription. In prokaryotes, regulation is focused on adjusting the cell’s activities to the environment to ensure viability. In multicellular eukaryotes, regulation is geared to maintaining internal homeostasis, and even in unicellular forms, this control has mechanisms to deal with a bounded nucleus and multiple chromosomes. ■■

How do evolutionary pressures for prokaryotes differ from those for multicellular eukaryotes?

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16 Control of Gene Expression 319

   16.2

Vantage point

Regulatory Proteins

= Hydrogen bond donors = Hydrogen bond acceptors = Hydrophobic methyl group = Hydrogen atoms unable to form hydrogen bonds

Learning Outcomes

DNA molecule 1

1. Explain how proteins can interact with base-pairs without unwinding the helix. 2. Describe the common features of DNA-binding motifs.

The ability of regulatory proteins to bind to specific DNA sequences and either block transcription or facilitate the binding of RNA polymerase is the basis for transcriptional control. To understand how cells control gene expression, it is first necessary to gain a clear picture of this molecular recognition process.

Major groove

H N

H N

Phosphate

G

N

H

N

N

H H

C N

N

O

H

H

It is not actually necessary for the DNA helix to be unwound for regulatory proteins to be able to interact with specific sequences of bases. When a protein binds to the surface of DNA, the edges of the bases are exposed in the two grooves in the molecule. The nature of the helix produces two grooves in the cylindrical structure of the helix: a larger major groove, and a smaller minor groove (see figure 14.8 for review). Within the deeper major groove, the nucleotides’ hydrogen bond donors and acceptors are accessible. The pattern created by these chemical groups is unique for each of the four possible base-pair arrangements, providing a ready way for a protein nestled in the groove to read the sequence of bases (figure 16.1).

Minor groove

DNA molecule 2 Major groove

H

DNA-binding domains interact with specific DNA sequences

320 part III Genetic and Molecular Biology

H

N

Proteins can interact with DNA through the major groove

Protein–DNA recognition is critical to all DNA functions, including replication, gene expression, and the control of these activities. The number of proteins that can interact with DNA to affect gene expression is quite large, but determining the structure of a subset of these led to the discovery of a small number of protein motifs that actually bind to DNA. These motifs probably arose relatively early in evolution, and have been reused with alterations in a wide variety of different proteins (motifs and domains are discussed in chapter 3). These DNA-binding motifs share the property of interacting with specific sequences of bases, usually through the major groove of the DNA helix. DNA-binding motifs are the key structure within the DNAbinding domain of these proteins. This domain is a functionally distinct part of the protein necessary to bind to DNA in a sequencespecific manner. Regulatory proteins also need to be able to interact with the transcription apparatus, which is accomplished by a different regulatory domain. Note that two proteins that share the same DNA-binding domain do not necessarily bind to the same DNA sequence. The similarities in the DNA-binding motifs appear in their threedimensional structure, and not in the specific contacts that they make with DNA.

O

N

H

Phosphate

N

N Sugar

A

N

H

H

O

N

CH3 H

T N

N H

O

Minor groove

Figure 16.1 Reading the major groove of DNA. Looking down into the major groove of a DNA helix, we can see the edges of the bases protruding into the groove. Each of the four possible base-pair arrangements (two are shown here) extends a unique set of chemical groups into the groove, indicated in this diagram by differently colored circles. A regulatory protein can identify the base-pair arrangement by this characteristic signature.

Several common DNA-binding motifs are shared by many proteins A limited number of common DNA-binding motifs are found in a wide variety of different proteins. Four of the best known are detailed here to give the sense of how DNA-binding proteins interact with DNA.

The helix-turn-helix motif The most common DNA-binding motif is the helix-turn-­helix, constructed from two α-helical segments of the protein linked by a short, nonhelical segment, the “turn” (figure 16.2a). As the first motif recognized, the helix-turn-helix motif has since been identified in hundreds of DNA-binding proteins. A close look at the structure of a helix-turn-helix motif reveals how proteins containing such motifs interact with the major groove of DNA. The helical segments of the motif interact with one another, so that they are held at roughly right angles. When this motif is pressed against DNA, one of the helical segments (called the recognition helix) fits snugly in the major groove of the DNA molecule, and the other butts up against the outside of the DNA molecule, helping to ensure the proper positioning of the recognition helix. Most DNA-regulatory sequences recognized by helixturn-helix motifs occur in symmetrical pairs. Such sequences are bound by proteins containing two helix-turn-helix motifs separated by 3.4 nanometers (nm), the distance required for one turn of the DNA helix (figure 16.2a). Having two protein– DNA-binding sites doubles the zone of contact between protein and DNA and greatly strengthens the affinity between them.

The homeodomain motif A special class of helix-turn-helix motif, the homeodomain, plays a critical role in development in a wide variety of eukaryotic organisms, including humans. Homeotic mutations in Drosophila cause one body part to be replaced by another. Analysis of the affected genes led to the discovery of the sequence of 60 amino acids that form the

homeodomain. The most conserved part of the homeodomain contains a recognition helix of a helix-turn-helix motif. This was the first indication that developmental mechanisms are ancient, and shared across greater phylogenetic distance than once was thought. At the level of our genes, we are much closer to a fly than you might imagine.

The zinc finger motif A different kind of DNA-binding motif uses one or more zinc atoms to coordinate its binding to DNA. Called zinc fingers, these motifs exist in several forms. In one form, a zinc atom links an α-helical segment to a β-sheet segment so that the helical segment fits into the major groove of DNA. This sort of motif often occurs in clusters, the β sheets spacing the helical segments so that each helix contacts the major groove. The effect is like a hand wrapped around the DNA with the fingers lying in the major groove. The more zinc fingers in the cluster, the more the protein associates with DNA.

The leucine zipper motif In another DNA-binding motif, the name actually refers to a dimerization motif that allows different subunits of a protein to associate with the DNA. This so-called leucine zipper is created where a region on one subunit containing several hydrophobic amino acids (usually leucines) interacts with a similar region on the other subunit. This interaction holds the subunits together and creates a 𝖸-shaped structure where the two arms of the 𝖸 are helical regions that each fit into the major groove of DNA but on opposite sides of the helix (figure 16.2b), holding the DNA like a pair of tongs. The Leucine Zipper Motif

The Helix-Turn-Helix Motif

α Helix (Recognition helix)

Turn

α Helix

Turn

Zipper region

α Helix

3.4 nm

90°

a.

b.

Figure 16.2 Major DNA-binding motifs. Two different DNA-binding motifs are pictured interacting with DNA. a. The helix-turn-helix motif binds to DNA using one α helix, the recognition helix, to interact with the major groove. The other helix positions the recognition helix. Proteins with this motif are usually dimers, with two identical subunits, each containing the DNA-binding motif. The two copies of the motif (red) are separated by 3.4 nm, precisely the spacing of one turn of the DNA helix. b. The leucine zipper acts to hold two subunits in a multisubunit protein together, thereby allowing α-helical regions to interact with DNA. chapter

16 Control of Gene Expression 321

Learning Outcomes Review 16.2

A DNA helix exhibits a major groove and a minor groove; regulatory proteins interact with DNA by accessing bases along the major groove. These proteins all contain DNA-binding motifs, and they often include one or two α-helical segments. These motifs form the active part of the DNA-binding domain, and another domain of the protein interacts with the transcription apparatus. ■■

What would be the effect of a mutation in a helix-turn-helix protein that altered the spacing of the two helices?

16.3 Prokaryotic

Regulation

Learning Outcomes 1. Contrast control by induction and control by repression. 2. Explain control of gene expression in the lac operon. 3. Explain control of gene expression in the trp operon.

The details of regulation can be revealed by examining mechanisms used by prokaryotes to control the initiation of transcription. Prokaryotes and eukaryotes share some common themes, but they have some profound differences as well. Later, in sections 16.4 and 16.5, we discuss eukaryotic systems and concentrate on how they differ from the simpler prokaryotic systems.

Activators are the logical and physical opposites of repressors. Effector molecules can either enhance or decrease activator binding.

Prokaryotes adjust gene expression in response to environmental conditions Changes in the environments that bacteria and archaea encounter often result in changes in gene expression. In general, genes encoding proteins involved in catabolic pathways (breaking down molecules) respond oppositely from genes encoding proteins involved in anabolic pathways (building up molecules). In the discussion that follows, we describe enzymes in the catabolic pathway that transports and utilizes the sugar lactose. Later, in this section, we will discuss the anabolic pathway that synthesizes the amino acid tryptophan. As mentioned in chapter 15, prokaryotic genes are often organized into operons, multiple genes that are part of a single transcription unit having a single promoter. Genes that are involved in the same metabolic pathway are often organized in this fashion. The proteins necessary for the utilization of lactose are encoded by the lac operon, and the proteins necessary for the synthesis of tryptophan are encoded by the trp operon.

?

Inquiry question What advantage might a bacterium

gain by linking several genes into a single operon, if all of the gene products participate in a single biochemical pathway?

Induction and repression

Control at the level of transcription initiation can be either positive or negative. Positive control increases the frequency of initiation, and negative control decreases the frequency of initiation. Each of these forms of control is mediated by regulatory proteins, but the proteins have opposite effects.

If a bacterium encounters lactose, it begins to make the enzymes necessary to utilize lactose. When lactose is not present, however, there is no need to make these proteins. Thus, we say that the synthesis of the proteins is induced by the presence of lactose. Induction therefore occurs when enzymes for a certain pathway are produced in response to a substrate. When tryptophan is available in the environment, a bacterium will not synthesize the enzymes necessary to make tryptophan. If tryptophan ceases to be available, then the bacterium begins to make these enzymes. Repression occurs when bacteria capable of making biosynthetic enzymes do not produce them. In the case of both induction and repression, the bacterium is adjusting to produce the enzymes that are optimal for its immediate environment.

Negative control by repressors

Negative control

Negative control is mediated by proteins called repressors. Repressors are proteins that bind to regulatory sites on DNA called operators to prevent or decrease the initiation of transcription. They act as a kind of roadblock to prevent the polymerase from initiating effectively. Repressors do not act alone; each responds to specific effector molecules. Effector binding can alter the conformation of the repressor to either enhance or abolish its binding to DNA. These repressor proteins are allosteric proteins with an active site that binds DNA and a regulatory site that binds effectors. Effector binding at the regulatory site changes the ability of the repressor to bind DNA (see chapter 6 for more details on allosteric proteins).

Control of the initiation of transcription can be either positive or negative. On the surface, repression may appear to be negative and induction positive, but this is not the case. In fact, regulation of both the lac and the trp operons are negatively controlled by repressor proteins. This seemingly opposite behavior is possible because the two repressor proteins interact with DNA and their respective effectors in the opposite way. For either mechanism to work, the molecule in the environment, such as lactose or tryptophan, must produce the proper effect on the gene being regulated. In the case of lac induction, the presence of lactose must prevent a repressor protein from binding to its regulatory sequence. In the case of trp repression, by contrast, the presence of tryptophan must cause a repressor protein to bind to its regulatory sequence. These responses are opposite because the needs of the cell are opposite in anabolic versus catabolic pathways. Each pathway is examined in detail to show how protein–DNA interactions allow the cell to respond to environmental conditions.

Control of transcription can be either positive or negative

Positive control by activators Positive control is mediated by another class of regulatory, allosteric proteins called activators that can bind to DNA and stimulate the initiation of transcription. These activators enhance the binding of RNA polymerase to the promoter to increase the frequency of transcription initiation. 322 part III Genetic and Molecular Biology

The lac operon is negatively regulated by the lac repressor

Gene for repressor protein Promoter for I gene

The control of gene expression in the lac operon was elucidated by the pioneering work of Jacques Monod and Fran ois Jacob. The lac operon consists of the genes that encode functions necessary to utilize lactose: β-galactosidase (lacZ), lactose permease (lacY), and lactose transacetylase (lacA), plus the regulatory regions necessary to control the expression of these genes (­figure 16.3). In addition, the gene for the lac repressor (lacI) is linked to the rest of the lac operon and is thus considered part of the operon although it has its own promoter. The arrangement of the control regions upstream of the coding region is typical of most prokaryotic operons, although the linked repressor is not.

Promoter for lac operon

CAP Plac

I

PI

CAP-binding site Operator

O

Gene for permease

Gene for β-galactosidase

Gene for transacetylase

Z

Y

Regulatory region

A

Coding region lac control system

Figure 16.3 The lac region of the Escherichia coli chromosome. 

The lac operon consists of a promoter, an operator, and three genes (lac Z, Y, and A) that encode proteins required for the metabolism of lactose. In addition, there is a binding site for the catabolite activator protein (CAP), which affects RNA polymerase binding to the promoter. The I gene encodes the repressor protein, which can bind the operator and block transcription of the lac operon.

the promoter (figure 16.4a). This binding prevents RNA polymerase from binding to the promoter. This DNA binding is sensitive to the presence of lactose: the repressor binds DNA in the absence of lactose, but does not in the presence of lactose.

Action of the repressor Initiation of transcription of the lac operon is controlled by the lac repressor. The repressor binds to the operator, which is adjacent to

Lactose Absent lac Operon Is Repressed

mRNA

lac repressor polypeptide CAP–binding site

lac repressor (No lactose present)

cAMP lac repressor

lac repressor CAP gene Promoter for lac operon RNA polymerase is blocked by the lac repressor

No transcription Enzymes to metabolize lactose not produced Z

Y

DNA

A

Operator

a. Lactose Present lac Operon Is Induced Allolactose (inducer) (lactose present)

mRNA

β-Galactosidase Translation

Transacetylase Permease

lac repressor cannot bind to DNA

RNA polymerase is not blocked and transcription can occur

mRNA

Z

Y

Enzymes to metabolize lactose produced

A

b.

Figure 16.4 Induction of the lac operon. a. In the absence of lactose the lac repressor binds to DNA at the operator site, thus

preventing transcription of the operon. When the repressor protein is bound to the operator site, the lac operon is shut down (repressed). b. The lac operon is transcribed (induced) when CAP is bound and when the repressor is not bound. Allolactose binding to the repressor alters the repressor’s shape so it cannot bind to the operator site and block RNA polymerase activity.

Data analysis What would the phenotype be of a repressor mutation that prevents DNA binding? Inducer binding? How do these compare with a mutation in the operator that prevents repressor binding? chapter

16 Control of Gene Expression 323

Interaction of repressor and inducer In the absence of lactose, the lac repressor binds to the operator, and the operon is repressed (figure 16.4a). The effector that controls the DNA binding of the repressor is a metabolite of lactose, allolactose, which is produced when lactose is available. Allolactose binds to the repressor, altering its conformation so that it no longer can bind to the operator (figure 16.4b). The operon is now induced. Since allolactose allows induction of the operon, it is usually called the inducer. As the level of lactose falls, allolactose concentrations d­ ecrease, making it no longer available to bind to the repressor and allowing the repressor to bind to DNA again. Thus this system of negative control by the lac repressor and its inducer, allolactose, allows the cell to respond to changing levels of lactose in the environment. Even in the absence of lactose, the lac operon is expressed at a very low level. When lactose becomes available, it is transported into the cell and enough allolactose is produced that induction of the operon can occur.

The presence of glucose prevents induction of the lac operon Glucose repression is a mechanism for the preferential use of glucose in the presence of other sugars such as lactose. If bacteria are

grown in the presence of both glucose and lactose, the lac operon is not induced. As glucose is used up, the lac operon is induced, allowing lactose to be used as an energy source. Despite the name glucose repression, this mechanism involves an activator protein that can stimulate transcription from multiple catabolic operons, including the lac operon. This activator, catabolite activator protein (CAP), is an allosteric protein with cAMP as an effector. This protein is also called cAMP response protein (CRP) because it binds cAMP, but we will use the name CAP to emphasize its role as a positive regulator. CAP alone does not bind to DNA, but binding of the effector cAMP to CAP changes its conformation such that it can bind to DNA (figure 16.5). The level of cAMP in cells is reduced in the presence of glucose so that no stimulation of transcription from CAPresponsive operons takes place. The CAP–cAMP system was long thought to be the sole mechanism of glucose repression. But more recent research has indicated that the presence of glucose inhibits the transport of lactose into the cell. This deprives the cell of the lac operon inducer, allolactose, allowing the repressor to bind to the operator. This mechanism, called inducer exclusion, is now thought to be the main form of glucose repression of the lac operon.

Glucose Low, Inducer Present, Promoter Activated DNA

Allolactose

cAMP–CAP binds to DNA

CAP

cAMP

Glucose level is low, cAMP is high

Repressor will not bind to DNA

CAPbinding site

mRNA

cAMP

Z

cAMP activates CAP by causing a conformation change

Y

A

RNA polymerase is not blocked and transcription can occur

a. Glucose High, Inducer Absent, Promoter Not Activated

Figure 16.5 Effect of glucose on the lac operon. Expression of the lac operon is controlled

324 part III Genetic and Molecular Biology

Repressor binds to DNA

A

CAP does not bind

Y

by a negative regulator (repressor) and a positive regulator (CAP). The action of CAP is sensitive to glucose levels. a. For CAP to bind to DNA, it must bind to cAMP. When glucose levels are low, cAMP is abundant and binds to CAP. The CAP–cAMP complex causes the DNA to bend around it. This brings CAP into contact with RNA polymerase (not shown), making polymerase binding to the promoter more efficient. b. High glucose levels produce two effects: cAMP is scarce so CAP is unable to activate the promoter, and the transport of lactose is blocked (inducer exclusion).

Glucose is available cAMP level is low

Z

Effector site is empty, and there is no conformation change

b.

RNA polymerase is blocked by the lac repressor

Given that inducer exclusion occurs, the role of CAP in the absence of glucose seems superfluous. But in fact, the positive control of CAP–cAMP is necessary because the promoter of the lac operon alone is not efficient in binding RNA polymerase. This inefficiency is overcome by the action of the positive control of the CAP–cAMP activator. Thus, the highest levels of expression occur in the absence of glucose and in the presence of lactose. In this case the presence of the activator and the absence of the repressor combine to produce the highest levels of expression (figure 16.5).

In the case of the trp operon these enzymes are necessary for synthesizing tryptophan. The regulatory region that controls transcription of these genes is located upstream of the genes. The trp operon is controlled by a repressor encoded by a gene located outside the trp operon. The trp operon is continuously expressed in the absence of tryptophan and is not expressed in the presence of tryptophan. The trp repressor is a helix-turn-helix protein that binds to the operator site located adjacent to the trp promoter (­figure  16.6). In the absence of tryptophan, the trp repressor does not bind to its operator, allowing expression of the operon and production of the enzymes necessary to make tryptophan.

The trp operon is controlled by the trp repressor Like the lac operon, the trp operon consists of a series of genes that encode enzymes involved in the same biochemical pathway.

Tryptophan Absent, Operon Derepressed E Inactive trp repressor (No tryptophan present)

D Translation

C B

mRNA

trp repressor cannot bind to DNA

Operator

Gene for trp repressor

A

E

Promoter for trp operon

D

C

B

Enzymes for tryptophan synthesis produced

A

RNA polymerase is not blocked, and transcription can occur

a. Tryptophan Present, Operon Repressed Tryptophan binds to repressor, causing a conformation change

Tryptophan

Repressor conformation change allows it to bind to the operator RNA polymerase is blocked by the trp repressor, and transcription cannot occur

E

D

C

B

A

Enzymes for tryptophan synthesis not produced

Gene for trp repressor

b.

Figure 16.6 How the trp operon is controlled. The tryptophan operon encodes the enzymes necessary to synthesize tryptophan.

a. The tryptophan repressor alone cannot bind to DNA. The promoter is free to function, and RNA polymerase transcribes the operon. b. When tryptophan is present, it binds to the repressor, altering its conformation so it now binds DNA. The tryptophan–repressor complex binds tightly to the operator, preventing RNA polymerase from initiating transcription. chapter

16 Control of Gene Expression 325

When levels of tryptophan rise, then tryptophan (the c­ orepressor) binds to the repressor and alters its confor­ mation, allowing it to bind to its operator. Binding of the repressor–­corepressor complex to the operator prevents RNA polymerase from binding to the promoter. The actual change in repressor structure due to tryptophan binding is an alteration of the orientation of a pair of helix-turn-helix motifs that allows their recognition helices to fit into adjacent major grooves of the DNA (figure 16.7). When tryptophan is present and bound to the repressor and this complex is bound to the operator, the operon is said to be repressed. As tryptophan levels fall, the repressor alone cannot bind to the operator, allowing expression of the operon. In this state, the operon is said to be ­derepressed, distinguishing this state from induction (see figure 16.6). The key to understanding how both induction and repression can be due to negative regulation is knowledge of the behavior of repressor proteins and their effectors. In induction, the repressor alone can bind to DNA, and the inducer prevents DNA binding. In the case of repression, the repressor only binds DNA when bound to the corepressor. Induction and repression are excellent examples of how i­nteractions of molecules can affect their structures, and how molecular structure is critical to function.

Learning Outcomes Review 16.3

Induction occurs when expression of genes in a pathway is turned on in response to a substrate; repression occurs when expression is prevented in response to a substrate. The lac operon is negatively controlled by a repressor protein that binds to DNA, thus preventing transcription. When lactose is present, the operon is turned on; allolactose binds to the repressor, which then no longer binds to DNA. This operon is also positively regulated by an activator protein. The trp operon is negatively controlled by a repressor protein that must be bound to tryptophan in order to bind to DNA. In the absence of tryptophan, the repressor cannot bind DNA, and the operon is derepressed. ■■

What would be the effect on regulation of the trp operon of a mutation in the trp repressor that can still bind to trp, but no longer bind to DNA?

16.4 Eukaryotic

Regulation

Learning Outcomes 1. Distinguish between the roles of general and specific transcription factors. 2. Describe the formation of a Pol II initiation complex. 3. Explain how transcription factors can have an effect from a distance in the DNA.

The control of transcription in eukaryotes is much more complex than in prokaryotes. The basic concepts of protein–DNA interactions are still valid, but the nature and number of interacting proteins are much greater due to some obvious differences. First, eukaryotes have their DNA organized into chromatin, complicating protein–DNA interactions considerably. Second, eukaryotic transcription occurs in the nucleus, and translation occurs in the cytoplasm; in prokaryotes, these processes are spatially and temporally coupled. This provides more opportunities for regulation in eukaryotes than for regulation in prokaryotes.

Tryptophan

Transcription factors can be either general or specific 3.4 nm

Figure 16.7 Tryptophan binding alters repressor conformation. The binding of tryptophan to the repressor increases the distance between the two recognition helices in the repressor, allowing the repressor to fit snugly into two adjacent portions of the major groove in DNA. 326 part III Genetic and Molecular Biology

In chapter 15, we introduced the concept of transcription factors. Eukaryotic transcription requires a variety of these protein factors, which fall into two categories: general transcription factors and specific transcription factors. General ­factors are necessary for the assembly of a transcription apparatus and recruitment of RNA polymerase II to a promoter. Specific factors increase the level of transcription in certain cell types or in response to signals.

General transcription factors Transcription of RNA polymerase II templates (mainly genes that encode protein products) requires more than just RNA polymerase II to initiate transcription. A host of general transcription factors are also necessary to recruit RNA polymerase II to a promoter and assemble an initiation complex for productive initiation. These factors are required for transcription to occur, but they do not increase the rate above this basal rate. General transcription factors are named with letter designations that follow the abbreviation TFII, for “transcription factor RNA polymerase II” (also true of polymerase I and III factors, except that these would be named TFI and TFIII, respectively). These general factors interact with other accessory factors and function to recognize a promoter. The RNA polymerase II enzyme interacts with the general factors to form an initiation complex that is competent to initiate transcription.

Specific transcription factors Specific transcription factors act in a tissue- or time-­dependent manner to stimulate higher levels of transcription than the basal level. The number and diversity of these factors are overwhelming. Some sense can be made of this proliferation of factors by concentrating on the DNA-binding motif, as opposed to the specific factors. Analysis of these specific transcription factors, called activators, revealed that they have two protein domains. One is a DNA-binding domain, and the other an activating domain that interacts with the transcription apparatus. These domains function independently in the protein. The independence of these domains was revealed by experiments that used recombinant DNA to “swap” the DNA-binding domains between two different factors. The recombinant proteins have their DNAbinding specificity switched, but both can still activate transcription.

Promoters and enhancers are binding sites for transcription factors Promoters are the binding sites for general transcription factors. A number of elements are found in promoters, but not all are found in all promoters. Thus, there is a “core promoter” consisting of a number of conserved elements, but not all promoters have all elements. The most common and most ancient element, the TATA box, is recognized by the TATA-binding protein, which is part of the TFIID factor. Another factor, TFIIB, binds to the TFIIB recognition element (BRE), adjacent to the TATA box in the core promoter, and interacts with RNA polymerase. An initiation complex is formed by the binding of TFIID, followed by binding of TFIIE, TFIIF, TFIIA, TFIIB, and TFIIH and a host of accessory factors called transcription-associated factors, TAFs. The initiation complex that results (figure 16.8) is much more complex than the bacterial equivalent, yet even this complex is only capable of basal levels of transcription; high levels require the action of other specific factors.

RNA polymerase II

Transcribed region

TAFs F

B TFIID

E

H TATA box A Core promoter

Figure 16.8 Formation of a eukaryotic initiation complex. The general transcription factor, TFIID, binds to the

TATA box and is joined by the other general factors, TFIIE, TFIIF, TFIIA, TFIIB, and TFIIH. This complex is added to by a number of transcription-associated factors (TAFs) that together recruit the RNA Pol II molecule to the core promoter.

Enhancers were originally defined as DNA sequences necessary for high levels of transcription that can act independently of position or orientation. This was counterintuitive to molecular biologists conditioned by prokaryotic systems to expect control regions to be immediately upstream of the coding region. We now know that enhancers are the binding sites for specific transcription factors, and their ability to act over large distances is due to DNA bending to form a loop to bring the enhancer closer to the promoter. Although more important in eukaryotic systems, this looping was first demonstrated using prokaryotic DNA-binding proteins (figure 16.9). This actually fits well with the emerging view of the organization of chromatin in the nucleus (see chapter 10). Topologically associated domains (TADs) are structures formed by chromosome loops that bring together physically distant regions on chromosomes. These TADs appear to correlate with regions that are being actively transcribed, and would allow an activator bound to an enhancer to be in close contact with transcription factors bound to a distant promoter (figure 16.10). It is likely that this kind of chromatin organization is much more important than physical distance on a chromosome.

Coactivators and mediators link transcription factors to RNA polymerase II Other factors specifically mediate the action of transcription factors. These coactivators and mediators are also necessary for activation of transcription by the transcription factor. They act by binding to the transcription factor and then binding to another part of the transcription apparatus. Mediators are essential to the function of some transcription factors, but not

chapter

16 Control of Gene Expression 327

Activator

Transcription factors

RNA polymerase Transcribed region Promoter

Enhancer

TATA box

5 nm NtrC (activator) Enhancer Promoter

ATP

mRNA synthesis RNA polymerase Bacterial RNA polymerase is loosely bound to the promoter. The activator (NtrC) binds at the enhancer.

ADP

Figure 16.10 How enhancers work. The enhancer site is located far away from the gene being regulated. Binding of an activator (gray) to the enhancer allows the activator to interact with the transcription factors (blue) associated with RNA polymerase, stimulating transcription.

5 nm DNA loops around so that the activator comes into contact with the RNA polymerase.

RNA polymerase

Activator mRNA synthesis

The activator triggers RNA polymerase activation, and transcription begins. DNA unloops.

Figure 16.9 DNA looping caused by proteins. When the bacterial activator NtrC binds to an enhancer, it causes the DNA to loop over to a distant site where RNA polymerase is bound, thereby activating transcription. Although such enhancers are rare in prokaryotes, they are common in eukaryotes. Courtesy of Dr. Harrison Echols and Dr. Sydney Kustu

328 part III Genetic and Molecular Biology

all ­transcription factors require them. The number of coactivators is much smaller­than the number of transcription factors because the same co­ac­ti­va­tor can be used with multiple transcription factors.

The transcription complex brings things together Although a few general principles apply, nearly every eukaryotic gene—or group of genes with coordinated regulation—is a unique case. Virtually all genes that are transcribed by RNA polymerase II need the same suite of general factors to assemble an initiation complex, but the assembly of this complex and its ultimate level of transcription depend on specific transcription factors that in combination make up the transcription complex (figure 16.11). The assembly of this transcription complex is also affected by the organization of chromatin in the nucleus, and regulators that affect chromatin structure. The organization of chromosomes into TADs can bring distant regions in close proximity, and create regions of active chromatin. The complexity of this organization, and the number of actors necessary, provide great flexibility in how cells can respond to the many signals a cell may receive affecting transcription, allowing integration of these signals.

Enhancers Coding region Activator Activators

General factors Enhancer Coactivator Activator

These regulatory proteins bind to DNA at distant sites known as enhancers. When DNA folds so that the enhancer is brought into proximity with the initiation complex, the activator proteins interact with the complex to increase the rate of transcription.

TAFs F

B

E

TFIID

RNA polymerase II

Coactivators These transcription factors stabilize the transcription complex by bridging activator proteins with the complex.

H

A

Core promoter

General Factors These transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to initiate transcription.

Figure 16.11 Interactions of various factors within the transcription complex. All specific transcription factors bind to enhancer sequences that may be distant from the promoter. These proteins can then interact with the initiation complex by DNA looping to bring the factors into proximity with the initiation complex. As detailed in the text, some transcription factors, called activators, can directly interact with the RNA polymerase II or the initiation complex, whereas others require additional coactivators.

?

Inquiry question How do eukaryotes coordinate the

activation of many genes whose transcription must occur at the same time?

Learning Outcomes Review 16.4

In eukaryotes, initiation requires general transcription factors that bind to the promoter and recruit RNA polymerase II to form an initiation complex. General factors produce the basal level of transcription. Specific transcription factors, which bind to enhancer sequences, can increase the level of transcription. Enhancers can act at a distance because DNA can loop, bringing an enhancer and a promoter closer together. Additional coactivators and mediators link certain specific transcription factors to RNA polymerase II. ■■

What would be the effect of a mutation that resulted in the loss of a general transcription factor versus the loss of a specific factor?

16.5 Chromatin

Structure Affects Gene Expression

Learning Outcomes 1. Describe at least two kinds of epigenetic mark. 2. Explain the function of chromatin-remodeling complexes.

In a diploid human cell, about 6 × 109 bp of DNA is wrapped into 3 × 107 nucleosomes. Further compaction forms the chromatin that fits about 2 m of DNA into a 5- to 10-μm nucleus. This obviously complicates the process of transcription. The way the cell deals with this conundrum is to selectively modulate the structure of chromatin by modifying DNA and histones, to allow the DNA transactions necessary for life. These alterations to chapter

16 Control of Gene Expression 329

chromatin are thought to be the basis for epigenetics––heritable changes in phenotype not due to changes in DNA sequence.

regulator polycomb repressive complex 2 (PRC2). This leads to the modification of histones associated with inactive chromatin.

Methylation of DNA, modification of histones, and noncoding RNA all affect chromatin structure

Histone modifications

One definition of an epigenetic alteration is that it must persist in the absence of the initiating stimulus, and be inherited through cell division. Alterations to chromatin structure can satisfy this definition, although the mechanisms of mitotic inheritance are not yet clear. Chromatin structure is affected by a wide variety of modifications to histones as well as DNA methylation, and even noncoding RNAs.

DNA methylation DNA methylation was the first modification of chromosome structure shown to act epigenetically. The addition of a methyl group to cytosine by a methylase enzyme creates 5-methylcytosine, but this change has no effect on its base-pairing with guanine (figure 16.12). High levels of DNA methylation correlate with inactive genes, and the allelespecific gene expression seen in genomic imprinting (see chapter 13) is mediated at least in part by DNA methylation. Methylation satisfies the stringent definition of epigenetic effector through well-known mechanisms. The semiconservative replication of DNA methylated on both strands produces hemimethylated DNA, which becomes fully methylated by the action of a maintenance methylase. In humans, DNMT1 is the maintenance methylase, and other methylases can initiate the process, but do not need to remain active.

The modification of histones is a complex topic as there are a large number of possible modifications, and four possible histones that can be modified. Modifications include acetylation and methylation of lysine; and phosphorylation of serine, threonine, and tyrosine. In general, acetylation, especially of H3, is correlated with active sites of transcription, both in regulatory regions and in the transcribed region of the gene itself. But methylation of the same histone (H3) can have the opposite effect, depending on the lysine methylated.

Some transcription activators alter chromatin structure The control of eukaryotic transcription utilizes the action of many different factors to activate transcription. Some activators interact directly with the initiation complex or with coactivators that interact with the initiation complex, as described in section 16.4. But in other cases, coactivators are enzymes called histone acetylases (HATs). This acetylation can increase transcription by altering higher-order chromatin structure that prevents transcription (figure 16.13). Some corepressors have been shown to function as histone deacetylases (HDACs), which remove acetyl groups from histones, as well. This led to the idea of a “histone code,” analogous to the genetic code. This histone code is postulated to affect chromatin structure, and thus to affect access to DNA by the transcription machinery. Nucleosomes can block the binding of RNA polymerase II to the promoter

X-chromosome inactivation Mammalian females inactivate one X chromosome as a form of dosage compensation to equalize X-chromosome expression in both sexes. First proposed by Mary Lyon in 1961, this continues to be an area of active research today. This is also the first example of a long noncoding RNA acting to regulate gene expression. The region of the chromosome that initiates the inactivation process contains a gene called X-inactivation-specific transcript (Xist), which does not encode a protein. The process of inactivation is quite complex, but a streamlined version is that Xist “coats” the entire inactive X chromosome, and acts to recruit the chromatin

Amino acid histone tail Condensed solenoid N-terminus Addition of acetyl groups to histone tails remodel the solenoid so that DNA is accessible for transcription

H N

H

Phosphate

O

N

N

G

H

H

CH3

N

N

H

C N

N N

H

O

H

Figure 16.12 DNA methylation. Cytosine is methylated,

creating 5-methylcytosine. Because the methyl group (green) is positioned to the side, it does not interfere with the hydrogen bonds of a G–C base-pair, but it can be recognized by proteins. 330 part III Genetic and Molecular Biology

Acetyl group

DNA available for transcription

Figure 16.13 Histone modification affects chromatin structure. DNA in eukaryotes is organized first into nucleosomes

and then into higher-order chromatin structures. The histones that make up the nucleosome core have amino tails that protrude. These amino tails can be modified by the addition of acetyl groups. The acetylation alters the structure of chromatin, making it accessible to the transcription apparatus.

Chromatin-remodeling complexes also change chromatin structure While the details of higher-order chromatin structure are not clear, it is clear that this structure can affect gene expression. So-called chromatin-remodeling complexes contain enzymes that modify histones and DNA, and alter chromatin structure directly. One class of remodeling factors are the ATP-dependent chromatin-remodeling factors. These function as molecular motors that use energy from ATP hydrolysis to alter the relationships between histones and DNA. They can catalyze four different changes in histone/DNA binding (figure 16.14): (1) nucleosome sliding along DNA, which changes the position of a nucleosome on the DNA; (2) creation of a remodeled state where DNA is more accessible; (3) removal of nucleosomes from DNA; and (4) replacement of histones with variant histones. These functions all make DNA more accessible to regulatory proteins that in turn affect gene expression. Chromatin is also modified during transcription elongation. The CTD domain of RNA polymerase II recruits a histone chaperone complex called FACT (facilitates chromatin transcription) that helps to remove nucleosomes from in front of the polymerase and reposition them behind the polymerase.

ATP

ADP + Pi

ATP-dependent remodeling factor

1. Nucleosome sliding

3. Nucleosome displacement

2. Remodeled nucleosome

4. Histone replacement

Figure 16.14 Function of ATP-dependent remodeling factors. ATP-dependent remodeling factors use the energy from

ATP to alter chromatin structure. They can (1) slide nucleosomes along DNA to reveal binding sites for proteins; (2) create a remodeled state of chromatin where the DNA is more accessible; (3) completely remove nucleosomes from DNA; and (4) replace histones in nucleosomes with variant histones.

Learning Outcomes Review 16.5

Eukaryotic DNA is packaged into chromatin, adding a structural challenge to transcription. Changes in chromatin structure correlate with modification of DNA and histones, and appear to be the basis for epigenetic changes. Some transcriptional activators modify histones by acetylation. Large chromatinremodeling complexes include enzymes that alter the structure of chromatin, making DNA more accessible to regulatory proteins. ■■

Why might some genes need to be expressed at all times?

16.6 Eukaryotic

Posttranscriptional Regulation

Learning Outcomes 1. Explain how small RNAs can affect gene expression. 2. Differentiate between the different kinds of posttranscriptional regulation.

The separation of transcription in the nucleus and translation in the cytoplasm in eukaryotes provides possible points of regulation that do not exist in prokaryotes. For many years we thought of this as “alternative” forms of regulation, but it now appears that they play a much more central role than previously suspected. In this section we will consider several of these mechanisms for controlling gene expression, beginning with the exciting new area of regulation by small RNAs.

Small RNAs act after transcription to control gene expression Developmental genetics has provided important insights into the regulation of gene expression. A striking example is the discovery of small RNAs that affect gene expression. The mutant lin-4 was known to alter developmental timing in the worm C. elegans, and genetic studies had shown that lin-4 regulated another gene, lin-14. When Ambros, Lee, and Feinbaum isolated the lin-4 gene in 1992, they found it did not encode a protein product. Instead, the lin-4 gene encoded only two small RNA molecules, one of 22 nt and the other of 61 nt. Furthermore, the 22-nt RNA was derived from the longer 61-nt RNA. Further work showed that this small RNA was complementary to a region in lin-14. A model was developed in which the lin-4 RNA acted as a translational repressor of the lin-14 mRNA (­figure 16.15). Although not called that at the time, this was the first identified microRNA, or miRNA. A completely different line of inquiry involved the use of double-stranded RNAs to turn off gene expression. This has been shown to act via another class of small RNA called small interfering RNAs, or siRNAs. These may be experimentally introduced, derived from invading viruses, or even encoded in the genome. The use of siRNA to control gene expression revealed the existence of cellular mechanisms for the control of gene expression via small RNAs. chapter

16 Control of Gene Expression 331

miRNA genes

SCIENTIFIC THINKING Hypothesis: The region of the lin-14 gene complementary to the lin-4 miRNA controls lin-14 expression. Prediction: If the lin-4 complementary region of the lin-14 gene is spliced into a reporter gene, then this reporter gene should show regulation similar to that of lin-14. Test: Recombinant DNA is used to make two versions of a reporter gene (β-galactosidase). In transgenic worms (C. elegans), expression of the reporter gene produces a blue color. 1. The β-galactosidase gene with the lin-14 3ʹ untranslated region

containing the lin-4 complementary region (construction shown below) 2. The β-galactosidase gene with a control 3ʹ untranslated region that lacks a lin-4 complementary region (not shown) Coding region

lin-4 complementary

lin-14 gene

β -Galactosidase gene β -Galactosidase with lin-14 3′ untranslated

miRNA biogenesis and function

Coding region

L1 Larva

L2 Larva

lin-14 3ʹ untranslated

Control 3ʹ untranslated Result: 1. Transgenic worms carrying reporter gene plus lin-14 3ʹ untranslated region show expression in L1 but not L2 stage larvae. This is the pattern expected for the lin-14 gene, which is controlled by lin-4. 2. Transgenic worms with control reporter gene with 3ʹ untranslated region that lacks lin-4 complementary region show expression in both L1 and L2 larvae. Conclusion: The 3ʹ untranslated region from lin-14 is sufficient to turn off gene expression in L2 larvae. Further Experiments: What expression pattern would you predict for these constructs in a mutant that lacks lin-4 function?

Figure 16.15 Control of lin-14 gene expression. The lin-14 gene is controlled by the lin-4 gene. This is mediated by a region of the 3' untranslated region of the lin-14 mRNA that is complementary to lin-4 miRNA. Since its discovery, gene silencing by small RNAs has been a source of great interest, both for its experimental uses and as an explanation for posttranslational control of gene expression. Research has uncovered a wealth of new types of small RNAs, but we will confine ourselves to the two classes of miRNA and siRNA, as these are well established and illustrate the RNA silencing machinery. 332 part III Genetic and Molecular Biology

The role of miRNAs in gene expression initially appeared to be confined to nematodes because the lin-4 gene did not have any obvious homologues in other systems. Seven years later, a second gene, let-7, was discovered in the same pathway in C. elegans. The let-7 gene also encoded a 22-nt RNA that could influence translation. In this case, homologues for let-7 were immediately found in both Drosophila and humans. As an increasing number of miRNAs were discovered in different organisms, miRNA gene discovery has turned to computer searching and high-throughput methods such as microarrays and new next-generation sequencing. A database devoted to miRNAs currently lists 1881 known human miRNA sequences. Genes for miRNA are found in a variety of locations, including the introns of expressed genes, and they are often clustered with multiple miRNAs in a single transcription unit. They are also found in regions of the genome that were previously considered transcriptionally silent. This finding is particularly exciting because other work looking at transcription across animal genomes has found that much of what we thought was transcriptionally silent is actually not. The production of a functional miRNA begins in the nucleus and ends in the cytoplasm with an approximately 22-nt RNA that functions to repress gene expression (figure 16.16). The initial transcript of a miRNA gene occurs by RNA polymerase II, producing a transcript called the pri-miRNA. The region of this transcript containing the miRNA can fold back on itself and base-pair to form a stem-andloop structure. This is cleaved in the nucleus by a nuclease called Drosha that trims the miRNA to just the stem-and-loop structure, which is now called the pre-miRNA. This pre-miRNA is exported from the nucleus through a nuclear pore bound to the protein exportin 5. Once in the cytoplasm, the pre-miRNA is further cleaved by another nuclease called Dicer to produce a short double-stranded RNA containing the miRNA. The miRNA is loaded into a complex of proteins called an RNA-induced silencing complex, or RISC. The RISC includes the RNA-binding protein Argonaute (Ago), which interacts with the miRNA. The complementary strand either is removed by a nuclease or is removed during the loading process. At this point, the RISC is targeted to repress the expression of other genes based on sequence complementarity to the miRNA. The complementary region is usually in the 3′ untranslated region of genes, and the result can be cleavage of the mRNA or inhibition of translation. It appears that in animals, the inhibition of translation is more common than the cleavage of the mRNA, although the precise mechanism of this inhibition is still unclear. In plants, the cleavage of the mRNA by the RISC is common and seems to be related to the complementarity found between plant miRNAs and their targets, which is more precise than that found in animal systems.

RNA interference Small RNA-mediated gene silencing has been known for a number of years. Some confusion arose in the nomenclature in this area because work in different systems led to a profusion of names. However, RNA interference, cosuppression, and posttranscriptional gene silencing all act through similar biochemical mechanisms.

Exogenous dsRNA, transposon, virus RNA polymerase II microRNA gene Repeated cutting by dicer Pri-microRNA

Nucleus

P

P

P

P

P

Pre-microRNA

Drosha

siRNAs

P

P

P

Exportin 5 siRNA in RISC

Ago

+

RISC Cytoplasm

Ago RISC

mRNA Dicer Ma Mature miRNA

Cleavage of target mRNA

Figure 16.17 Biogenesis and function of siRNA. SiRNAs Ago RISC

mRNA

Ago RISC

can arise from a variety of sources that all produce long doublestranded regions of RNA. The double-stranded RNA is processed by Dicer nuclease to produce a number of siRNAs that are each loaded onto their own RISC. The RISC then cleaves target mRNA.

mRNA RNA cleavage

Target mRNA

Ago

Ago

RISC

RISC

bition of translation translattion Inhibition

Figure 16.16 Biogenesis and function of miRNA. 

Genes for miRNAs are transcribed by RNA polymerase II to produce a pri-miRNA. This is processed by the Drosha nuclease to produce the pre-miRNA, which is exported from the nucleus bound to export factor Exportin 5. Once in the cytoplasm, the pre-miRNA is processed by Dicer nuclease to produce the mature miRNA. The miRNA is loaded into a RISC, which can act either to cleave target mRNAs, or to inhibit translation of target mRNAs.

The term RNA interference is currently the most commonly used and involves the production of siRNAs. The production of siRNAs is similar to that of miRNAs, except that they arise from a long piece of double-stranded RNA (­ figure  16.17). This can be either a very long region of self complementarity, or from two complementary RNAs. These long double-stranded RNAs are processed by Dicer to yield multiple siRNAs that are loaded into an Ago containing RISC.

The siRNAs usually have near-perfect complementarity to their target mRNAs, and the result is cleavage of the mRNA by the siRNA containing RISC. The double-stranded RNA used to produce an siRNA can originate inside a cell or be introduced from outside the cell. From the cell itself, genes can produce RNAs with long regions of self-complementarity that fold back to produce a substrate for Dicer in the cytoplasm. They can also arise from repeated regions of the genome that contain transposable elements. Exogenous double-stranded RNAs can be introduced experimentally or by infection with a virus.

Small RNAs may have evolved to protect the genome The observation that viral RNA can be degraded via the RNAsilencing pathway may point toward the evolutionary origins of small RNAs. A related observation is that RNA silencing can control the action of transposons as well. In both mice and fruit flies, genetic evidence supports the involvement of the RNA interference machinery in the germ line where a specific class of small RNA appears to be involved in silencing transposons during spermatogenesis and oogenesis. Thus the origins of this mechanism may be an ancient pathway for protection of the genome from assault from both within and without. The conservation of key chapter

16 Control of Gene Expression 333

proteins suggests that the ancestor to all eukaryotes had some form of RNA-silencing pathway.

Distinguishing miRNAs and siRNAs The biogenesis of both miRNA and siRNA involves cleavage by Dicer and incorporation into a RISC complex. The main thing that distinguishes these small RNAs is their targets: miRNAs tend to repress genes different from their origin, whereas endogenous siRNAs tend to repress the genes they were derived from. Additionally, siRNAs are used experimentally to turn off the expression of genes. This technique uses the cells’ RNA-silencing machinery to turn off a gene by introducing a double-stranded RNA complementary to the gene. The two classes of small RNA have other differences. When multiple species are examined, miRNAs tend to be evolutionarily conserved, but siRNAs do not. Although the biogenesis for both is similar in terms of the nucleases involved, the actual structure of the double-stranded RNAs is not the same. The transcript of miRNA genes form stem-loop structures containing the miRNA, but the double-stranded RNAs generating siRNAs may be bimolecular, or very long stem-loops. These longer double-stranded regions lead to multiple siRNAs, whereas only a single miRNA is generated from a pre-miRNA.

Small RNAs can mediate heterochromatin formation RNA-silencing pathways have also been implicated in the formation of heterochromatin in fission yeast, plants, and ­Drosophila. In fission yeast, centromeric heterochromatin formation is driven by siRNAs produced by the action of the Dicer nuclease. This heterochromatin formation also involves modification of histone proteins and thus connects RNA interference with chromatin-remodeling complexes in this system. It is not yet clear how widespread this phenomenon is. Plants are an interesting case in that they have a variety of small RNA species. The RNA interference pathway in plants is more complex than that in animals, with multiple forms of Dicer 1

1

2

2

3

nuclease proteins and Argonaute RNA-binding proteins. One class of endogenous siRNA can lead to heterochromatin formation by DNA methylation and histone modification.

Alternative splicing can produce multiple proteins from one gene The high frequency of alternative splicing discussed in chapter 15 indicates over 200,000 transcripts produced from the human genome. Elucidating the functional significance of this large number of transcripts will require analysis of specific genes. The pattern of splicing can change during different stages of development or in different tissues. We will briefly consider two examples. An example of alternative splicing that produces different developmental outcomes occurs in Drosophila. Sex determination in Drosophila is the result of a complex series of alternative splicing events that differ between males and females. A human example of tissue-specific alternative splicing involves proteins produced by the thyroid gland and the hypothalamus. These two organs produce two distinct hormones: calcitonin and CGRP (calcitonin-gene-related peptide) from one gene. Calcitonin controls calcium uptake and the balance of calcium in tissues such as bones and teeth. CGRP is involved in a number of neural and endocrine functions. Despite their different physiological roles, they are produced from the same primary transcript (figure 16.18). Whether calcitonin or CGRP is produced depends on different splicing factors in the thyroid and the hypothalamus. A functional analysis of every gene in the human genome is unrealistic at present. Another approach is to analyze many proteins at the same time using high-throughput mass spectrometry. This technology is evolving quickly, and this proteomics approach holds great promise. Early studies have produced conflicting results as to how many transcripts are translated.

RNA editing alters mRNA after transcription In some cases, the editing of mature mRNA transcripts can produce an altered mRNA that is not truly encoded in the ­genome—an

3

4

4

5

5

6 3′ Poly-A tail

5′ cap Primary RNA transcript Exons Introns 1

2

Introns are spliced

Thyroid splicing pattern 3

4

1 3′ Poly-A tail

5′ cap

Hypothalamus splicing pattern 2

3

5′ cap

5

6 3′ Poly-A tail

Mature mRNA

Mature mRNA

Calcitonin

CGRP

Figure 16.18 Alternative splicing. Many primary transcripts can be spliced in different ways to give rise to multiple mRNAs. In this example, in the thyroid the primary transcript is spliced to contain four exons encoding the protein calcitonin. In the hypothalamus the fourth exon, which contains the poly-A site used in the thyroid, is skipped and two additional exons are added to encode the protein calcitonin-gene-related peptide (CGRP). 334 part III Genetic and Molecular Biology

unexpected possibility. RNA editing was first discovered as the insertion of uracil residues into some RNA transcripts in protozoa, and it was thought to be an anomaly. RNA editing of a different sort has since been found in mammalian species, including humans. In this case, the editing involves chemical modification of a base to change its base-pairing properties, usually by deamination. For example, both deamination of cytosine to uracil and deamination of adenine to inosine have been observed (inosine pairs as G would during translation).

The 5-HT serotonin receptor RNA editing has been observed in some brain receptors for opiates in humans. One of these receptors, the serotonin (5-HT) receptor, is edited at multiple sites to produce a total of 12 different isoforms of the protein. It is unclear how widespread RNA editing is, but it is further evidence that the information encoded within genes is not the end of the story for protein production.

mRNA is transported out of the nucleus Processed mRNA transcripts exit the nucleus through the nuclear pores (described in chapter 4). The passage of a transcript across the nuclear membrane is an active process that requires the transcript to be recognized by receptors lining the interior of the pores. Specific portions of the transcript, such as the poly-A tail, appear to play a role in this recognition. There is little hard evidence that gene expression is regulated at this point, although it could be. On average, about 10% of primary transcripts consist of exons that will make up mRNA sequences, but only about 5% of the total mRNA produced as primary transcript ever reaches the cytoplasm. This observation suggests that about half of the exons in primary transcripts never leave the nucleus, but it is unclear whether the disappearance of this mRNA is selective.

Initiation of translation can be controlled The translation of an mRNA by ribosomes in the cytoplasm requires a complex of proteins called translation factors. In some cases, gene expression is regulated by modification of one or more of these factors. In other instances, translation repressor proteins shut down translation by binding to the beginning of the mRNA, preventing ribosomes from binding to the mRNA. In humans, the production of ferritin (an iron-storing protein) is normally shut off by a translation repressor protein called aconitase. Aconitase binds to a 30-nt sequence at the beginning of the ferritin mRNA, forming a stable loop to which ribosomes cannot bind. When iron enters the cell, it binds to aconitase, causing the aconitase to dissociate from ferritin mRNAs, allowing them to be translated. This increases ferritin production 100-fold.

The degradation of mRNA is controlled The level of gene expression also depends on how long mRNA persists in the cytoplasm. Unlike prokaryotic mRNAs, which typically have a half-life of about 3 min, eukaryotic mRNAs are very stable. For example, β-globin gene transcripts have a half-life of over 10 hr, an eternity in the fast-moving metabolic life of a cell.

The mRNAs encoding regulatory proteins and growth factors are usually much less stable, with half-lives of less than 1 hr. What makes these particular transcripts so unstable? Many of these mRNAs have specific sequences near their 3′ end that make them targets for RNA-degrading enzymes. A sequence of A and U nucleotides near the 3′ poly-A tail of a transcript promotes removal of the tail. Loss of the poly-A tail leads to rapid degradation by 3′- to 5′-RNA exonucleases. Loss of the poly-A tail also stimulates decapping enzymes that remove the 5′ cap, leading to degradation by 5′- to 3′-RNA exonucleases. Other short-lived mRNAs have sequences near their 3′ ends that are recognized by endonucleases, which cause these transcripts to be digested quickly. It is important for mRNAs encoding regulatory proteins to have a short half-life to allow cells to rapidly alter the levels of regulatory proteins. Another control on the level of mRNAs is called nonsensemediated decay (NMD). This is a mechanism for removing mRNAs with a premature termination signal (nonsense codon). This prevents the production of truncated proteins due to premature termination. Many alternative splice variants produce early termination codons, and are thus removed in the cytoplasm.

Learning Outcomes Review 16.6

Small RNAs control gene expression by either selective degradation of mRNA, inhibition of translation, or alteration of chromatin structure. Multiple mRNAs can be formed from a single gene via alternative splicing, which can be tissue- and developmentally specific. The sequence of an mRNA transcript can also be altered by RNA editing. ■■

How could the phenomenon of RNA interference be used in drug design?

16.7 Protein

Degradation

Learning Outcomes 1. Describe the role of ubiquitin in the degradation of proteins. 2. Explain the function of the proteasome.

If all of the proteins produced by a cell during its lifetime remained in the cell, serious problems would arise. Protein labeling studies in the 1970s indicated that eukaryotic cells turn over proteins in a controlled manner. That is, proteins are continually being synthesized and degraded. Although this protein turnover is not as rapid as in prokaryotes, it indicates that a system regulating protein turnover is important. Proteins can become altered chemically, rendering them nonfunctional; in addition, the need for any particular protein may be transient. Proteins also do not always fold correctly, or they may become improperly folded over time. These changes can lead to loss of function or other chemical behaviors, such as aggregating into insoluble complexes. In fact, a number of neurodegenerative diseases, such as Alzheimer dementia, Parkinson chapter

16 Control of Gene Expression 335

Ubiquitin Protein to be destroyed ATP

ADP + Pi

Destroyed by proteolysis

Figure 16.19 Ubiquitination of proteins. Proteins that are to be degraded are marked with ubiquitin. The enzyme ubiquitin ligase uses ATP to add ubiquitin to a protein. When a series of these have been added, the polyubiquitinated protein is destroyed.

disease, and mad cow disease, are related to proteins that aggregate, forming characteristic plaques in brain cells. Thus, in addition to normal turnover of proteins, cells need a mechanism to get rid of old, unused, and incorrectly folded proteins. Enzymes called proteases can degrade proteins by breaking peptide bonds, converting a protein into its constituent amino acids. Although there is an obvious need for these enzymes, they clearly cannot be floating around in the cytoplasm active at all times. One way that eukaryotic cells handle such problems is to confine destructive enzymes to a specific cellular compartment. You may recall from chapter 4 that lysosomes are vesicles that contain digestive enzymes, including proteases. Lysosomes are used to remove proteins and old or nonfunctional organelles, but this system is not specific for particular proteins. Cells need another regulated pathway to remove proteins that are old or unused, but leave the rest of cellular proteins intact.

Addition of ubiquitin marks proteins for destruction Eukaryotic cells solve this problem by marking proteins for destruction, then selectively degrading them. The mark that cells use is the attachment of a ubiquitin molecule. Ubiquitin, so named because it is found in essentially all eukaryotic cells (that is, it is ubiquitous), is a 76-amino-acid protein that can exist as an isolated molecule or in longer chains that are attached to other proteins. The longer chains are added to proteins in a stepwise fashion by an enzyme called ubiquitin ligase (figure 16.19). This reaction requires ATP and other proteins, and it takes place in a multistep, regulated process. Proteins that have a ubiquitin chain attached are called polyubiquitinated, and this state is a signal to the cell to destroy this protein. Two basic categories of proteins become ubiquitinated: those that need to be removed because they are improperly folded or nonfunctional, and those that are produced and degraded in a controlled fashion by the cell. An example of the latter are the cyclin proteins that help to drive the cell cycle (see chapter 10). When 336 part III Genetic and Molecular Biology

15 nm

Figure 16.20 The Drosophila proteasome. The central complex contains the proteolytic activity, and the flanking regions act as regulators. Proteins enter one end of the cylinder and are cleaved to peptide fragments that exit the other end. ©Image of PDB ID 4B4T

(Unverdorben P, et al., “Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome,” PNAS (2014) 111: 5444-5549). ©2015 David Goodsell & RCSB Protein Data Bank (www.rcsb.org). Molecule of the Month DOI 10.2210/rcsb_pdb/mom_2013_10

these proteins have fulfilled their role in active division of the cell, they become polyubiquitinated and are removed. In this way, a cell can control entry into cell division or maintain a nondividing state.

The proteasome degrades polyubiquitinated proteins The macromolecular machine in the cell that degrades proteins marked with ubiquitin is the proteasome, a large cylindrical complex that proteins enter at one end and from which they exit at the other as amino acids or peptide fragments (figure 16.20).

Degradation

Polypeptide fragments Proteasome

ATP ADP +

Ubiquitination

Pi

Ubiquitin ADP +

Pi

ATP

ATP Targeted protein

ADP

Ubiquitin ligase

Figure 16.21 Degradation by the ubiquitin– proteasome pathway. Proteins are first ubiquitinated, then enter

the proteasome to be degraded. In the proteasome, the polyubiquitin is removed and then is later “deubiquitinated” to produce single ubiquitin molecules that can be reused.

The proteasome complex contains a central region that has protease activity and regulatory components at each end. Although not a membrane-bounded organelle, this macromolecular machine is a form of compartmentalization on a very small scale. In a twostep process, first to mark proteins for destruction, then to process them through a large complex, proteins to be degraded are isolated from the rest of the cytoplasm. The process of ubiquitination followed by degradation by the proteasome is called the ubiquitin–proteasome pathway. It can be

RNA polymerase II DNA 3′

5′

Primary RNA transcript

thought of as a cycle in that the ubiquitin added to proteins is not itself destroyed in the proteasome. As the pro­­teins are degraded, the ubiquitin chain itself is simply cleaved back into ubiquitin units that can then be reused (figure 16.21). A review of various methods of the control of eukaryotic gene expression is provided in figure 16.22.

1. Initiation of transcription Access to DNA by regulatory proteins depends on chromatin structure. Initiation of transcription is controlled by transcription factors that bind promoters and enhancers.

?

Inquiry question What are two reasons a cell would polyubiquitinate a polypeptide?

Cut intron

Exons Introns

5′ cap

3′ poly-A tail

Mature RNA transcript

Large subunit

3′ poly-A tail

Nuclear pore

mRNA Small subunit

4. Protein synthesis Many proteins take part in the translation process, and regulation of the availability of any of them alters the rate of gene expression by speeding or slowing protein synthesis.

5′

Protein

6. Protein degradation Proteins to be degraded are labeled with ubiquitin, then destroyed by the proteasome.

3. Passage through the nuclear membrane Gene expression can be regulated by controlling access to or efficiency of transport channels.

5′ cap

3′

Ubiquitin

2. RNA splicing Gene expression can be controlled by altering the rate of splicing in eukaryotes. Alternative splicing can produce multiple mRNAs from one gene.

5. RNA interference Gene expression is regulated by small RNAs. Protein complexes containing siRNA and miRNA target specific mRNAs for destruction or inhibit their translation.

RISC

Proteasome

Figure 16.22 Mechanisms for control of gene expression in eukaryotes. chapter

16 Control of Gene Expression 337

Learning Outcomes Review 16.7

Control of protein degradation in eukaryotes involves addition of the protein ubiquitin, which marks the protein for destruction. The proteasome, a cylindrical complex with protease activity in its center, recognizes ubiquitinated proteins and breaks them down,

much as a shredder destroys documents. Ubiquitin is recycled unchanged. ■■

If the ubiquitination process were not tightly controlled, what effect would this have on a cell?

Chapter Review Science Source

16.1 Control of Gene Expression Control can occur at all levels of gene expression. Transcription is controlled by regulatory proteins that modulate the ability of RNA polymerase to bind to the promoter. These may either block transcription or stimulate it.

The presence of glucose prevents induction of the lac operon. Maximal expression of the lac operon requires positive control by catabolite activator protein (CAP) complexed with cAMP. When glucose is low, cAMP is high. Glucose repression involves both inducer exclusion, in which lactose is prevented from entering the cell, and the control of CAP function by the level of glucose.

Control strategies in eukaryotes maintain homeostasis and drive development.

The trp operon is controlled by the trp repressor. The trp operon is repressed when tryptophan, acting as a corepressor, binds to the repressor, altering its conformation such that it can bind to DNA and turn off the operon. This prevents expression in the presence of excess trp.

16.2 Regulatory Proteins

16.4 Eukaryotic Regulation

Proteins can interact with DNA through the major groove. A DNA double helix exhibits a major groove and a minor groove; bases in the major groove are accessible to regulatory proteins.

Transcription factors can be either general or specific. General transcription factors are needed to assemble the transcription apparatus and recruit RNA polymerase II at the promoter. Specific factors act in a tissue- or time-dependent manner to stimulate higher rates of transcription.

Control strategies in prokaryotes are geared to adjust to environmental changes.

DNA-binding domains interact with specific DNA sequences. A region of the regulatory protein that can bind to the DNA is termed a DNA-binding motif (figure 16.2). Several common DNA-binding motifs are shared by many proteins. Common motifs include the helix-turn-helix motif, the homeodomain motif, the zinc finger motif, and the leucine zipper.

16.3 Prokaryotic Regulation Control of transcription can be either positive or negative. Negative control is mediated by proteins called repressors that interfere with transcription. Positive control is mediated by a class of regulatory proteins called activators that stimulate transcription. Prokaryotes adjust gene expression in response to environmental conditions. The lac operon is induced in the presence of lactose; that is, the enzymes to utilize lactose are produced only when lactose is present. The trp operon is repressed; that is, the enzymes needed to produce tryptophan are turned off when tryptophan is present. The lac operon is negatively regulated by the lac repressor. The lac operon is induced when the effector (allolactose) binds to the repressor, altering its conformation such that it no longer binds DNA (figure 16.4). 338 part III Genetic and Molecular Biology

Promoters and enhancers are binding sites for transcription factors. General factors bind to the promoter to recruit RNA polymerase. Specific factors bind to enhancers, which may be distant from the promoter but can be brought closer by DNA looping. Coactivators and mediators link transcription factors to RNA polymerase II (figure 16.11). Some, but not all, transcription factors require a mediator. The number of coactivators is small because a single coactivator can be used with multiple transcription factors. The transcription complex brings things together.

16.5 Chromatin Structure Affects Gene Expression The packaging of DNA into nucleosomes complicates transcription. Cells can alter chromatin structure by modifying DNA or histones. This is thought to be the basis for epigenetics. Methylation of DNA, modification of histones, and noncoding RNA all affect chromatin structure. Methylation of DNA bases, primarily cytosine, correlates with genes that have been “turned off.” X-chromosome inactivation involves a noncoding RNA and polycomb repressor complex.

Alternative splicing can produce multiple proteins from one gene. In response to tissue-specific factors, alternative splicing of pre-mRNA from one gene can result in multiple proteins.

Some transcription activators alter chromatin structure. Acetylation of histones results in active regions of chromatin. Chromatin-remodeling complexes also change chromatin structure. Chromatin-remodeling complexes contain enzymes that move, reposition, and transfer nucleosomes.

RNA editing alters mRNA after transcription. mRNA is transported out of the nucleus. Initiation of translation can be controlled. Translation factors may be modified to control initiation; translation repressor proteins can bind to the beginning of a transcript so that it cannot attach to the ribosome.

16.6 Eukaryotic Posttranscriptional Regulation Small RNAs act after transcription to control gene expression. RNA interference involves siRNAs formed by cleavage of doublestranded RNA by the Dicer nuclease. An siRNA binds an Argonaute protein to form an RNA-induced silencing complex (RISC), which can cleave mRNA or inhibit translation. An miRNA is formed by two nucleases, Drosha and Dicer. These also form a RISC that can either degrade mRNA or stop translation.

The degradation of mRNA is controlled. An mRNA transcript is relatively stable, but it may carry targets for enzymes that degrade it more quickly as needed by the cell.

16.7 Protein Degradation

Small RNAs may have evolved to protect the genome. Viral RNAs are degraded and transposons are silenced in the germ line by RNA interference. The origins of this machinery are ancient.

Addition of ubiquitin marks proteins for destruction. In eukaryotes, proteins targeted for destruction have ubiquitin added to them as a marker.

Small RNAs can mediate heterochromatin formation. In fission yeast, Drosophila, and plants, RNA interference pathways lead to the formation of heterochromatin.

The proteasome degrades polyubiquitinated proteins. A cellular machine—the cylindrical proteasome—degrades ubiquitinated proteins that pass through it.

Visual Summary requires

Control of gene expression in

Prokaryotic cell

Regulatory proteins

Environmental changes

respond to

Eukaryotic cell

involves

involves

contain Induction

Repression

lac operon

trp operon

Initiation complex

DNA binding domain include

Specific DNA sequences

negative regulation positive regulation +

Repressor

Activator

Inducer (lactose) ‒

lac repressor CAP activator

×

binds

Operator

Corepressor (tryptophan) +

‒

trp repressor binds

×

Operator

Chromatin structure

includes Transcription factors

involves

Mediator

Post-translational regulation includes

DNA methylation

Small RNAs

‘off’

Alternative splicing

either

General

Specific

bind

bind

Promoter

Enhancers

Histone acetylation ‘on’ Chromatin remodeling

chapter

Translation initiation

mRNA degradation

16 Control of Gene Expression 339

Review Questions Science Source

U N D E R S TA N D 1. In prokaryotes, control of gene expression usually occurs at the a. splicing of pre-mRNA into mature mRNA. b. initiation of translation. c. initiation of transcription. d. All of the choices are correct. 2. Regulatory proteins interact with DNA by a. b. c. d.

unwinding the helix and changing the pattern of base-pairing. binding to the sugar–phosphate backbone of the double helix. unwinding the helix and disrupting base-pairing. binding to the major groove of the double helix and interacting with base-pairs. 3. In E. coli, induction in the lac operon and repression in the trp operon are both examples of a. b. c. d.

negative control by a repressor. positive control by a repressor. negative control by an activator. positive control by an activator.

4. The lac operon is controlled by two main proteins. These proteins a. b. c. d.

both act in a negative fashion. both act in a positive fashion. act in the opposite fashion, one negative and one positive. act at the level of translation.

5. In eukaryotes, binding of RNA polymerase to a promoter requires the action of a. b. c. d.

specific transcription factors. general transcription factors. repressor proteins. inducer proteins.

6. In eukaryotes, the regulation of gene expression occurs a. b. c. d.

only at the level of transcription. only at the level of translation. at the level of transcription initiation, or posttranscriptionally. only posttranscriptionally.

7. In the trp operon, the repressor binds to DNA a. b. c. d.

in the absence of trp. in the presence of trp. in either the presence or the absence of trp. only when trp is needed in the cell.

A P P LY 1. The lac repressor, the trp repressor, and CAP are all a. b. c. d.

negative regulators of transcription. positive regulators of transcription. allosteric proteins that bind to DNA and an effector. proteins that can bind DNA or other proteins.

2. Specific transcription factors in eukaryotes interact with enhancers, which may be a long distance from the promoter. These transcription factors then a. b. c. d.

alter the structure of the DNA between enhancer and promoter. do not interact with the transcription apparatus. can interact with the transcription apparatus via DNA looping. can interact with the transcription apparatus by removing the intervening DNA.

340 part III Genetic and Molecular Biology

3. Repression in the trp operon and induction in the lac operon are both mechanisms that a. b. c. d.

would be possible only with positive regulation. allow the cell to control the level of enzymes to fit environmental conditions. would be possible only with negative regulation. cause the cell to make the enzymes from these two operons all the time.

4. Regulation by small RNAs and alternative splicing are similar in that both a. b. c. d.

act after transcription. act via RNA/protein complexes. regulate the transcription machinery. Both a and b are correct.

5. Eukaryotic mRNAs differ from prokaryotic mRNAs in that they a. b. c. d.

usually contain more than one gene. are collinear with the genes that encode them. are not collinear with the genes that encode them. Both a and c are correct.

6. In the cell cycle, cyclin proteins are produced in concert with the cycle. This likely involves a. b. c. d.

control of initiation of transcription of cyclin genes, and ubiquitination of cyclin proteins. alternative splicing of cyclin genes to produce different cyclin proteins. RNA editing to produce the different cyclin proteins. transcription/translation coupling.

7. A mechanism of control in E. coli not discussed in this chapter involves pausing of ribosomes, allowing a transcription terminator to form in the mRNA. In eukaryotic fission yeast, this mechanism should a. b. c. d.

be common since they are unicellular. not be common since they are unicellular. not occur as transcription occurs in the nucleus and translation in the cytoplasm. not occur due to possibility of alternative splicing.

SYNTHESIZE 1. You have isolated a series of mutants affecting regulation of the lac operon. All of these are constitutive, that is, they express the lac operon all the time. You also have both mutant and wild-type alleles for each mutant in all combinations, and on F ′ plasmids, which can be introduced into cells to make the cell diploid for the relevant genes. How would you use these tools to determine which mutants affect DNA binding sites on DNA, and which affect proteins that bind to DNA? 2. Examples of positive and negative control of transcription can be found in the regulation of expression of the bacterial operons lac and trp. Use these two operon systems to describe the difference between positive and negative regulation. 3. What forms of eukaryotic control of gene expression are unique to eukaryotes? Could prokaryotes use the mechanisms, or are they due to differences in these cell types? 4. The number and type of proteins found in a cell can be influenced by genetic mutation and regulation of gene expression. Discuss how these two processes differ.

17 CHAPTER

Biotechnology Chapter Contents 17.1

Recombinant DNA

17.2

Amplifying DNA Using the Polymerase Chain Reaction

17.3

Creating, Correcting, and Analyzing Genetic Variation

17.4

Constructing and Using Transgenic Organisms

17.5

Environmental Applications

17.6

Medical Applications

17.7

Agricultural Applications

0.3 μm Professor Stanley N. Cohen/Science Source

Introduction

Visual Outline includes

includes

Biotechology involves

DNA technology used to analyze

uses

Recombinant DNA

and

Polymerase chain reaction

include

include

for

Transgenic animals

Environmental applications

Transgenic plants

Molecular cloning

to Amplify DNA

Genetic variation

Applications

Transgenic organisms

5′

3′

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3′ 5′

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Medical applications

Agricultural applications

While it may appear futuristic, biotechnology has a long history. The use of yeast to produce leavened bread dates to ancient Egypt, and using fermentation to produce wine probably predates this by several thousand years. Modern biotechnology combines recent discoveries in molecular biology and genetics with more traditional biotechnologies such as selective breeding and hybridization. Modern biotechnology takes advantage of advances in a variety of disciplines. Progress in computer science accelerated genome sequencing, and discoveries in chemical engineering moved nanobiotechnology out of the realm of science fiction. In this chapter, we explore ­modern biotechnology by first considering advances in molecular biology and ­genetics such as the use of plasmids and other recombinant DNA techniques. Then, we consider how biotechnology can be used to solve socially relevant problems in environmental science, medicine, and agriculture.

How restriction endonucleases work

17.1 Recombinant

DNA

Learning Outcomes 1. Describe how restriction endonucleases and ligases are used to make recombinant DNA. 2. Explain how DNA fragments can be separated with gel electrophoresis and why this is useful. 3. Describe the construction and uses of recombinant DNA libraries.

Biotechnology is not necessarily a discipline of the 21st century. For example, the initial domestication of dogs from wolves by our hunter-gatherer ancestors some 15,000 years ago and their subsequent selective breeding have produced the 180 existing dog breeds. Later, our agriculturalist ancestors began to domesticate crops, and the development of hybrid corn in the early 20th century substantially increased yield. Recent discoveries in genetics and molecular biology have ushered in a modern era of biotechnological innovation and discovery. The ability to isolate and manipulate DNA revolutionized biotechnology, accelerated rates of discovery, and made possible novel applications of ­biotechnology. The construction of recombinant DNA, a single DNA molecule made from two different sources, began in the mid-1970s. In 1972, Paul Berg created the first recombinant DNA molecule when he inserted viral DNA into bacterial DNA. Building on Berg’s work in 1973, Herbert Boyer and Stanley Cohen introduced genes from the toad Xenopus laevis into bacteria and showed that the genes could be passed from generation to generation and were expressed in the bacteria. This conclusively demonstrated that the genes from slowly reproducing ­animals could be replicated and expressed in much more rapidly growing bacteria. In the following three decades, the discoveries of Berg, Boyer, and Cohen would become the basis of biotechnologies from whole-genome sequencing to the production of insulin in bacteria.

Restriction endonucleases cleave DNA at specific sites These early cloning experiments were some of the first to utilize new tools called restriction enzymes. Arising from basic research on how the host range of a bacterial virus can be restricted, these enzymes recognize specific sequences of DNA, and act as a ­nuclease to cleave the DNA. Originally identified by Werner ­Arbor studying the host restriction phenomenon, the first enzyme that cleaved DNA at its recognition site was isolated by Hamilton Smith from the bacterium Haemophilus influenza and thus named HindIII. Arbor, Smith, and Daniel Nathans shared the 1978 Nobel Prize in Physiology or Medicine for this work. The discovery of restriction endonucleases is important for two reasons: first, for their ability to cut DNA into specific fragments, and second, for their use in genome mapping (see chapter 18). 342 part III Genetic and Molecular Biology

There are three types of restriction enzymes, but only type II cleaves at precise locations, which enable creation of recombinant ­molecules. These enzymes recognize a specific DNA s­ equence, ranging from 4 bases to 12 bases, and cleave the DNA at a specific base within this sequence (figure 17.1). The recognition sites for most type II e­ nzymes are palindromes. A linguistic palindrome is a word or phrase that reads the same forward and in reverse, such as the name “Hanah.” The palindromic DNA ­sequence reads the same from 5′ to 3′ on one strand as it does on the complementary strand. Given this kind of sequence, cutting the DNA at the same base on either strand produces staggered cuts that leave “sticky ends,” or overhangs. These short, unpaired sequences are the same for any DNA that is cut by this enzyme. This allows DNA molecules from different sources to be easily joined together due to the short regions of complementarity in the overhangs (figure 17.1). Although less common, some type II restriction enzymes can cut both strands in the same position, producing blunt, not sticky, ends. Blunt-cut ends can be joined with other blunt-cut ends.

Gel electrophoresis separates DNA fragments To create a piece of recombinant DNA, two or more fragments of DNA must be joined together. Before this can happen, the fragments of DNA must be purified, or isolated. A common technique to separate different fragments of cut DNA so they can be isolated is gel electrophoresis. This technique takes advantage of the negative charge on DNA by using an electrical field to provide the force necessary to separate DNA molecules based on size. The gel, which is made of either agarose or polyacryl­amide and spread thinly on supporting material, provides a three-­ dimensional matrix that separates molecules based on size ­(figure 17.2). The gel is submerged in a buffer solution containing ions that can carry current and is subjected to an electrical field. The strong negative charges from the phosphate groups in the DNA backbone cause it to migrate toward the positive pole. The gel acts as a sieve to separate DNA molecules based on size: the larger the molecule, the more slowly it will move through the gel matrix. Over a given ­period, smaller molecules migrate farther than larger ones. The DNA in gels can be visualized using a fluorescent dye that binds to DNA. Gels can be made that are used solely to analyze the sizes of DNA fragments, or so that individual fragments of DNA can be cut from the gel, purified, and prepared for further use.

DNA fragments are combined to make recombinant molecules The next step in constructing a recombinant DNA molecule is to combine pieces of DNA from different sources into a single molecule. The recombinant DNA can be stably replicated in a host organism such as the common gut bacterium, Escherichia coli. The most common strategy to create a stable recombinant DNA molecule is to add a fragment of DNA of interest—for example, a gene sequence to be studied—into a bacterial plasmid. The recombinant plasmid can then be introduced to E. coli, where it will be replicated and maintained.

EcoRI

DNA duplex

EcoRI

Restriction sites

G

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EcoRI Restriction endonuclease cleaves the DNA.

EcoRI Restriction endonuclease cleaves the DNA.

A

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G

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C

Sticky ends

Sticky ends

A

G C

T

T

A

A

A

T

T

A

T

T

C

Figure 17.1 Many restriction endonucleases produce DNA fragments with sticky ends. The

restriction endonuclease EcoRI always cleaves the sequence 5′–GAATTC–3′ between G and A. Because the same sequence occurs on both strands, both are cut. However, the two sequences run in opposite directions on the two strands. As a result, single-stranded tails called “sticky ends” are produced that are complementary to each other. These complementary ends can then be joined to a fragment from another DNA that is cut with the same enzyme. These two molecules can then be joined by DNA ligase to produce a recombinant molecule.

G

A

DNA from another source cut with the same restriction endonuclease is added. A

A

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C G

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DNA ligase joins the strands.

Recombinant DNA molecule

Joining fragments of DNA using DNA ligase DNA fragments that have been cut by restriction endonucleases and purified using an agarose gel can be joined together using DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond between the 5′ phosphate group of one strand of DNA and the 3′ hydroxyl group of another strand of DNA. This is the same reaction that is used by the DNA ligase that joins Okazaki fragments together during lagging strand DNA synthesis in the cell (see chapter 14). If two fragments of cut DNA have sticky ends, then the short complementary overhangs (as shown in figure 17.1) will hold the two strands of DNA together while DNA ligase forms the phosphodiester bond. Certain DNA ligases can also join DNA fragments with blunt ends. In figure 17.3, a piece of foreign DNA is being introduced into plasmid that has been cut by a specific restriction endonuclease. The foreign DNA has been cut by the same restriction endonuclease and thus has ends that are complementary to the cut ends in the plasmid. The sticky ends will hold the two molecules together, and DNA ligase can form phosphodiester bonds, joining them to create a recombinant plasmid. The whole process of producing recombinant DNA molecules is often called molecular cloning.

Reverse transcriptase makes DNA from RNA Although the “central dogma” of molecular biology indicates that the flow of information is from DNA to RNA to protein, there are

several instances in biology when DNA can be made using the information in RNA. Reverse transcriptase, first identified in a class of viruses called retroviruses, can use RNA as a template to synthesize a DNA molecule (see chapter 26). Retroviruses, like the human immunodeficiency virus (HIV), have RNA genomes but then convert the RNA to DNA when they infect cells. Reverse transcriptase is an RNAdependent DNA polymerase, which means that it synthesizes DNA, but using RNA as a template. Reverse transcriptase is also a DNA-dependent DNA polymerase, which means it can also use DNA as a template to synthesize DNA. The DNA made from the information in mRNA is called complementary DNA (cDNA). Reverse transcriptase can be used to create cDNAs from mRNAs isolated from eukaryotic cells (figure 17.4). This allows the analysis of only sequences that are actually used to direct the synthesis of proteins.

DNA libraries are collections of recombinant DNA molecules A library is a collection of many different recombinant DNA molecules, from a single source, that can be stably maintained and replicated in a suitable host organism. The source could be an ­entire genome, or all of the cDNA produced by a specific cell type, or any population of DNA molecules you wish to analyze. ­Libraries are constructed by splicing together different DNA fragments of interest with a DNA molecule engineered to propagate chapter

17 Biotechnology 343

Restriction Enzyme Digestion

Gel Electrophoresis

DNA samples are cut with restriction enzymes in three different reactions producing fragments of different sizes.

Samples from the restriction enzyme digests are introduced into the gel. Electrical current is applied, causing fragments to migrate through the gel.

Restriction endonuclease 1 cut

Reaction Reaction Reaction 1 2 3

Power source

Reaction 1 200 bp

800 bp

Mixture of DNA fragments of different sizes in solution placed at the top of “lanes” in the gel Lane

Restriction endonuclease 2 cut

− Cathode

Reaction 2 400 bp

Gel

600 bp Restriction endonuclease 3 site

+

Reaction 3

Anode 900 bp

Buffer

100 bp

a.

b. Visualizing Stained Gel Gel is stained with a dye to allow the fragments to be visualized. bp 1517 1200 1000

L

1

2

3

Figure 17.2 Gel electrophoresis separates DNA fragments based on size. 

a. Three restriction enzymes are used to cut DNA into specific pieces, depending on each enzyme’s recognition sequence. b. The fragments are loaded into a gel (agarose or polyacrylamide), and an electrical current is applied. The DNA fragments migrate through the gel based on size, with larger fragments moving more slowly. c. This results in a pattern of fragments separated based on size, with the smaller fragments migrating farther than larger ones. A series of fragments of known sizes produces a ladder so that sizes of fragments of unknown size can be estimated (bp = base-pairs; L = ladder; 1, 2, 3 = fragments from piece of DNA cut with restriction endonucleases 1, 2, and 3, respectively).

500 100

c.

Figure 17.3 Molecular cloning with vectors. Plasmids are cut using restriction

DNA Ligase Joins DNA Molecules

Restriction endonuclease

Foreign DNA No DNA inserted

Ampicillin resistance gene

Restriction enzyme cuts recombinant plasmid.

344 part III Genetic and Molecular Biology

Foreign DNA and DNA ligase are added.

DNA inserted

endonucleases at a specific site, and foreign DNA and DNA ligase are added. If foreign DNA is successfully incorporated into the plasmid, a recombinant plasmid is created. In a process called transformation, the plasmids can be introduced into bacteria. Spreading transformed cells on a medium containing the antibiotic ampicillin selects for plasmid-containing cells.

exons introns

1

Plasmid Library 1

2

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Eukaryotic DNA template Transcription 5′ cap

3′ poly-A tail

Primary RNA transcript

DNA fragments from source DNA

DNA inserted into plasmid vector

Introns are cut out, and coding regions are spliced together. 5′ cap

3′ poly-A tail

Figure 17.5 Creating a plasmid library. Fragments of

DNA with the correct sticky ends can be joined with cut plasmid vectors so that each plasmid contains a different piece of DNA. Transformation of the recombinant plasmids into bacterial cells produces a library of bacteria with each bacterium containing plasmid with a unique DNA insert.

Transformation

Mature RNA transcript Isolation of mRNA Addition of reverse transcriptase Reverse transcriptase Reverse transcriptase utilizes mRNA to create cDNA. mRNA–cDNA hybrid Addition of mRNAdegrading enzymes Degraded mRNA

DNA polymerase

Double-stranded cDNA with no introns

Figure 17.4 Making cDNA from mRNA using reverse transcription. A mature mRNA transcript is usually much smaller

than the gene producing it due to the removal of introns. mRNA is isolated from the cytoplasm of a cell, which the enzyme reverse transcriptase uses as a template to make a DNA strand complementary to the mRNA. The newly made strand of DNA is the template for the enzyme DNA polymerase, which assembles a complementary DNA strand along it, producing cDNA—a double-stranded DNA version of the mature mRNA.

the library (figure 17.5). We call these specialized DNA molecules cloning vectors; they include plasmids and artificial ­chromosomes. The choice of which vector to use for a library depends on how it will be used, and on the size of the pieces of DNA in the library. Cloning vectors have several important features: 1. a sequence that allows replication in a host organism (such as E. coli or yeast); 2. a selectable marker, such as resistance to an antibiotic; and 3. sequences that allow DNA fragments to be added (restriction endonuclease or recombination sites).

Each cell contains a single fragment. All cells together are the library.

Once a library has been made, different techniques are used to identify and isolate specific recombinant molecules of interest. Libraries can be made that can produce proteins to be studied, for determining genome sequence and structure, or for isolating particular genes. A DNA library can be stored by being introduced into an appropriate host organism (figure 17.5). Growing the host cells will then “amplify” the library and produce many copies of ­recombinant molecules that can be isolated or analyzed. Libraries can be introduced into bacteria or yeast by transformation, a process where cells are treated to make them take up DNA from the ­environment. The host organism for the library depends on the cloning vector used and the purpose of the library. For small DNA fragments, bacterial plasmids work well and each bacterial cell in the library will contain an individual recombinant plasmid. When the cells are grown in culture, the cloned DNAs are passively replicated by DNA replication. For libraries with larger pieces of DNA (such as genomic fragments), bacterial or yeast artificial chromosomes are used and the DNA library is stored in bacteria or yeast.

Reverse transcriptase is used to make cDNA libraries Cells from a specific developmental time point, or a specific ­tissue, can be used to isolate mRNA to use as a template for cDNA synthesis (see figure 17.4). The resulting cDNAs can then be used to construct a library that represents only the genes e­ xpressed at a specific time point, or in a specific tissue. Genomic libraries made from different cells or tissues of an organism all contain similar sequences, but this will not be true for cDNA libraries. A brain cDNA library will contain many brain-specific sequences not ­represented in a fibroblast cDNA library, which in turn will c­ ontain fibroblast-specific sequences not represented in the brain cDNA library. chapter

17 Biotechnology 345

Because a collection of cDNAs made from a tissue reflects the mRNA present in that tissue, comparisons of the collections of cDNAs produced from different tissues can be informative. For example, comparisons made between cDNA libraries from different cell types might provide information about the relationships between the genes being expressed, the proteome, and cell structure and function.

Learning Outcomes Review 17.1

Type II restriction endonucleases cleave DNA at specific sites. Gel electrophoresis uses an electric field to separate DNA fragments according to size. DNA ligase will join DNA fragments from different sources to produce recombinant DNA. Reverse transcription converts mRNA into cDNA. DNA libraries are stable collections of recombinant DNA molecules used for many kinds of genetic analyses. ■■

Why is it important to be able to convert mRNA into DNA?

Data analysis The human genome contains 3 billion

base-pairs. If you construct a genomic library of the human genome with 500-bp fragments of DNA, what is the minimum number of clones in your library?

17.2 Amplifying

DNA Using the Polymerase Chain Reaction

Learning Outcomes 1. Relate the process of DNA replication to PCR. 2. Compare and contrast PCR, RT-PCR, and quantitative RT-PCR.

The development of the polymerase chain reaction (PCR) in 1993 accelerated the construction of recombinant DNA molecules and ushered in a new era of genetic engineering. PCR mimics the processes of DNA replication to produce millions of copies of a DNA sequence (a process called amplification) without the need for molecular cloning. A major benefit of producing DNA fragments using PCR is that the exact sequence of DNA to be amplified can be chosen. When DNA molecules are cloned into vectors based on producing DNA fragments by restriction endonuclease digestion, the DNA fragments available are limited by the location of restriction endonuclease sites. To amplify a DNA sequence using PCR, two 20- to 25nucleotide-long DNA sequences called primers are chemically synthesized. One primer is complementary to one strand of the DNA at one end of the chosen sequence, and the other primer is complementary to the opposite strand at the other end of the ­chosen sequence. When the primers bind to their complementary DNA 346 part III Genetic and Molecular Biology

sequences, DNA polymerase can extend the two primers so that two new strands of DNA are produced. These two strands of DNA are complementary and contain the original primer binding sites. Repeating this process cyclically results in an exponential increase in the number of DNA molecules. A DNA sequence up to about 10,000 base-pairs long can be amplified this way.

PCR mimics DNA replication PCR mimics the processes of DNA replication, and an individual reaction is cycled through a series of steps where each step is analogous to a step in DNA replication: 1. Denaturation. Heat is used to separate strands of doublestranded DNA. 2. Annealing of primers. Primers provide the 3′ OH required for elongation by DNA polymerase. 3. Synthesis. DNA polymerase makes new DNA. New DNA molecules are synthesized exponentially with repeated cycles; a 25-cycle PCR produces 225 molecules of DNA from a single target molecule! When PCR was initially developed, the steps were done manually using a mixture containing the DNA to be amplified (the template DNA), and the primers for the fragment to be amplified. The mixture was heated to 95°C to denature the DNA and then cooled to a temperature for primers to anneal to the template; then, a DNA polymerase was added for synthesis. Because the 95°C denaturation step also denatures the polymerase enzyme, new enzyme needed to be added with each cycle. As a typical PCR involves 25 to 30 cycles, this made PCR tedious and labor intensive. PCR was made practical with two innovations. First, a thermostable DNA polymerase called Taq polymerase was isolated from the thermophilic bacterium Thermus aquaticus. This overcame the need to add new DNA polymerase during each cycle because Taq polymerase is stable at the denaturation temperature. The second innovation was the development of machines with heating blocks that can be accurately and rapidly cycled over large temperature ranges. A typical PCR is now set up in a single tube containing the template DNA, the primers, nucleotides, and a thermostable DNA polymerase. Although Taq polymerase is still commonly used, other more accurate thermostable polymerases are also used. The tube is placed into a PCR machine that can quickly cycle between the denaturation temperature, the primer annealing temperature, and the DNA synthesis temperature (figure 17.6). Twenty-five cycles of PCR can be performed in as little as an hour. This has made PCR one of the most common techniques for generating specific DNA molecules for a variety of uses.

Reverse transcription PCR makes amplified DNA from mRNA In addition to using a variety of types of DNA (plasmids, genomic DNA) as a template, PCR can also be performed on cDNA made from mRNA. Isolated mRNA is incubated with reverse transcriptase, then the resulting cDNA is used as a template for PCR. The combination of these two techniques is called reverse transcription PCR (RT-PCR). RT-PCR is useful for three reasons. First, it allows

Figure 17.6 The polymerase chain reaction. 

DNA segment to be amplified 5′

3′

3′

5′

The polymerase chain reaction (PCR) allows a single DNA sequence to be amplified for analysis. The process involves using short primers for DNA synthesis that flank the region to be amplified and (1) repeated rounds of denaturation, (2) annealing of primers, and (3) synthesis of DNA. The enzyme used for synthesis is a thermostable DNA polymerase that can work at the high temperatures needed for denaturation of template DNA. The reaction is performed in a thermocycler machine that can be programmed to change temperatures quickly and accurately. The annealing temperature used depends on the length and base composition of the primers. Details of the synthesis process have been simplified here to illustrate the amplification process. Newly synthesized strands are shown in light blue with primers in green. If there was one DNA molecule to start, then at the end of cycle one there would be two molecules, at the end of cycle two, four molecules, and at the end of cycle three, eight molecules.

PCR machine

1. Sample is first heated to denature DNA. DNA is denatured into single strands 5′

3′

3′

5′

2. DNA is cooled to a lower temperature to allow annealing of primers. 5′

3′

Primers anneal to DNA 3′

5′

3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers. 5′ 3′

5′

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5′ Taq DNA polymerase 5′ 3′

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Quantitative RT-PCR can determine levels of an mRNA Cells often respond to changes in environmental conditions, both internal and external, with changes in gene expression. Analysis of gene expression involves accurate quantification of cellular mRNA levels. One of the fastest and easiest ways to measure relative changes in gene expression is using reverse transcription quantitative PCR (RT-qPCR). This involves isolating mRNA, using reverse transcriptase to convert this to cDNA, then using PCR to amplify specific cDNAs using gene-specific primers. The amount of DNA produced can be measured in real time by the PCR machine. For this reason, quantitative PCR is also known as real-time PCR. Two common techniques are used, both of which depend on the use of fluorescent dyes.

3′

3′ 5′

5′ 3′

3′

5′

5′ End of cycle 1 Two copies

the creation of recombinant DNA molecules containing DNA copies of only the exons of genes. Second, it allows study of the structure and function of gene products. Third, it can be used to determine relative levels of gene expression in cells and tissues.

End of cycle 2 Four copies

End of cycle 3 Eight copies

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DNA quantification using fluorescent DNA-binding dyes The simplest way to quantify the amount of DNA made during the PCR part of RT-qPCR is to add a dye to the PCR reaction that binds nonspecifically to DNA. If DNA is present, the dye will bind to the DNA and fluoresce when illuminated by a laser. After each cycle of PCR, a laser in the PCR machine illuminates the reaction and detectors measure the amount of fluorescence. As the amount of DNA increases with each PCR cycle, more dye becomes bound to the DNA, and the fluorescence increases. The data can be output to a computer to plot a graph of fluorescence versus the number of PCR cycles. Imagine two qPCR reactions using cDNA made from the same amount of mRNA isolated from two different cell types. During the qPCR part of the experiment, fluorescence might chapter

17 Biotechnology 347

increase in one of the samples faster than in the other. In the qPCR where fluorescence appeared faster, there was initially more mRNA for a specific gene because more DNA molecules were made as PCR proceeded. Computers can be used to quantify the relative differences between samples to investigate differences in gene expression under different conditions.

5′

b.

It took 13 years, from 1990 to 2003, to obtain a finished version of the sequence of the human genome at an estimated cost of $2.7 billion. Today, a human genome can be sequenced in a few days for around $1000. DNA sequencing requires that many copies of fragments of the genome be obtained. For the initial Human Genome Project, this involved molecular cloning of fragments of genomic DNA into vectors that were engineered to facilitate the manual DNA sequencing (see chapter 18). Clones then had to be grown in bacteria, and DNA isolated for sequencing, all of which is both labor intensive and time consuming. 348 part III Genetic and Molecular Biology

3′

5′

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PCR primer

Polymerization 3′

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3′ Probe displacement and cleavage

Fluorescence 3′

5′ PCR products

Result Cleavage products

Fluorescence

a. No DNA 1 pg 10 pg 100 pg 1 ng 10 ng 100 ng 1 μg

RFU

PCR is the basis of many sequencing technologies

Quencher

Probe Fluorophore

PCR primer

DNA quantification using fluorescent DNA-binding probes A more accurate way to quantify the amount of DNA produced by a PCR reaction is to introduce a fluorescently labeled probe into the qPCR reaction. This probe consists of a 20- to 30-nucleotidelong sequence of single-stranded DNA that is complementary to a portion of the DNA being analyzed. Probes are chemically synthesized, and can be modified for different applications. In qPCR, a probe complementary to a short region of the template cDNA has a fluorescent molecule added to one end. When illuminated with one wavelength of light, the fluorophore emits a different wavelength of light that can be measured by the PCR machine. A ­so-called quencher molecule is added to the other end of the probe. This quencher will absorb the light emitted by the fluorophore, thus preventing detection. As long as both fluorophore and ­quencher are attached to the same probe, fluorescence from the fluorophore is quenched. If the fluorophore and quencher are separated, then fluorescence will be detected. If the probe is included in the PCR reaction, it can be used to detect the amount of DNA produced. After the denaturation step, when the reaction is cooled to the primer annealing ­temperature, the qPCR probe also anneals to one of 16,000 the template strands between the two PCR primers. As the polymerase extends the PCR primers, it will run 14,000 into the annealed probe on one strand. As the Taq poly12,000 merase has an exonuclease activity (see chapter 14), it can degrade the probe and complete copying the 10,000 template. When the probe is degraded, the fluorophore 8,000 is released from the quencher, and its fluorescence can be detected (figure 17.7a). The PCR cycles continue 6,000 and with every round, there will be an increase in fluo4,000 rescence that is proportional to the amount of DNA being made. The more template (cDNA made from 2,000 mRNA) there is to begin with, the faster the fluorescent 0 signal will increase (figure 17.7b).

3′

0

5

10

15

20 25 Cycle Number

30

35

40

Figure 17.7 Quantification of mRNA levels by RT-qPCR.  a. Degradation of a fluorescent probe as amplification occurs results in an increase in fluorescence. b. As DNA is amplified, the amount of fluorescence increases proportionally to the number of molecules of DNA present. The more DNA that is present at the start of the reaction, the more quickly fluorescence appears during the PCR (pg, picograms; ng, nanograms).

PCR overcame the need to use cloning vectors to obtain enough DNA to be sequenced. Many new sequencing technologies, called “next-generation” sequencing (NGS), use variations of PCR to produce the DNA that will be sequenced (discussed in

chapter 18). Without PCR, sequencing genomes quickly and cheaply would not be possible.

PCR is used in diagnostic tests for many diseases Since infectious agents, be they viral, bacterial, or fungal, all contain nucleic acids, PCR can form the basis of relatively rapid and extremely sensitive diagnostic tools. All these tests require is a set of primers that will specifically amplify only nucleic acid from the pathogen of interest. For bacteria and DNA viruses, all you need are primers specific for some gene, or genes, in the pathogen’s genome not found in the human genome. Depending on the nature of the clinical sample used, this may not even require isolating DNA, as the denaturation temperature will break open most cells or viruses. For RNA viruses, it is more complicated, as it requires ­using RT-PCR, and handling RNA is more difficult. Most diagnostic kits used for RNA viruses begin with an RNA isolation step, using one of the commercial RNA isolation kits. The RNA is then ready for reverse transcription to produce cDNA, which is then used for the PCR reaction. This can be streamlined, but the basic outline is the same. One sidelight of the 2020 COVID-19 pandemic has been the development of more, faster, and better versions of these kinds of diagnostic kits.

Learning Outcomes Review 17.2

The polymerase chain reaction (PCR) is used to rapidly amplify specific DNA sequences from small amounts of starting material. RT-PCR allows DNA copies of expressed mRNAs to be amplified, and RT-qPCR allows the relative quantification of specific mRNAs. PCR is also used in next-generation sequencing and diagnostic kits for infectious agents. ■■

Individuals in sexually reproducing populations are phenotypically diverse, yet all members of a species usually have the same set of genes. Most diversity seen in populations is produced by complex interactions between genetic variation, epigenetic differences, and environmental variability. Advances in manipulating and analyzing DNA have produced new ways to study the genetic variation that contributes to phenotypic variation. Analyzing genetic variation is important because it contributes to understanding differences between individuals within a species, and between species. Recent advances in DNA technology allow us to create or repair mutant alleles as a way to investigate gene function.

Forensics uses DNA fingerprinting to identify individuals It is sometimes desirable to be able to identify an individual based on a small amount of tissue or bodily fluids. This occurs during the investigation of crimes, and in the identification of victims after catastrophic events. Although more sensitive techniques have been developed (see chapter 18), forensics continues to use DNA fingerprinting as a primary technique. This is at least in part due to the large databases that have been assembled by law enforcement organizations. DNA fingerprinting takes advantage of short, repeated sequences that vary among individuals. Short tandem repeats ­ (STRs), typically 2 to 4 nt long, are not part of coding or regulatory regions of genes and mutate over generations so that the length of the ­repeats varies. We say that the population is polymorphic for these molecular markers. A combination of these markers can be used as DNA “fingerprints” in criminal investigations and other identification applications (figure 17.8). This is also another example of the flexibility and utility of the PCR process. Primers are designed to flank a region known to

How are PCR and DNA replication similar? How do they differ? STRs and DNA fingerprinting

?

Inquiry question Suppose you wanted a copy of a section

of a eukaryotic genome that included the introns and exons. Would the creation of cDNA be a good way to go about this? Explain.

1 Control Ladder

2

3

4

5

6

7

DYS19 STR Y chromosome

bp 202 absent 198 194 190 186 178 absent

17.3 Creating,

Correcting, and Analyzing Genetic Variation

Learning Outcomes 1. Describe how genetic information can be used to identify an unknown individual. 2. Explain how we can construct mutations in genes in vitro. 3. Describe the pros and cons of RNA interference and direct genome editing.

172 168

D12S66 STR Chromosome 12

160 156 152 absent 148 absent

Figure 17.8 Using STRs and DNA fingerprinting to identify individuals. A Y-chromosome STR distinguishes between

men and women (top) because it is absent in women (column 2). The STR occurs in different lengths in men, depending on the number of repeats (columns 3–7). A second STR found on chromosome 12 appears in both men and women (bottom). Courtesy Lutz Roewer, Department of Forensic Genetics, Institute of Legal Medicine and Forensic Sciences, Charité - Universitätsmedizin Berlin

chapter

17 Biotechnology 349

contain an STR. These primers can then be used to amplify the STR-containing region from very small amounts of starting ­material. The analysis can also be done using the same technology used for automated sequencing (see chapter 18). Since 1997, 13 STRs have been established as the standard of evidence for identification in court, and approved identification kits are available commercially. The 13 STRs form the basis of a federal profiling database called CODIS (Combined DNA Index System). New DNA fingerprints can be compared with those of known individuals in CODIS. DNA fingerprinting has also been used to exonerate wrongly convicted individuals who had been imprisoned for years before DNA analysis was widely available, as well as to identify individuals after catastrophes. After the September 11, 2001, attacks on the World Trade Center in New York, DNA fingerprinting was the only means for identifying some of the victims. After a devastating earthquake in the Republic of Haiti in 2010, DNA fingerprinting was used to reunite children with their families.

PCR can be used to create point mutations in specific genes Depending on its location in the genome, a change in one or a few nucleotides may or may not have an effect on phenotype. Any change to the genome that results in a phenotypic change we recognize as a mutation. For years geneticists created mutations randomly using chemicals (mutagens), but now we can create mutations in vitro. Studying mutations allows us to better understand the normal function of the altered DNA. The ultimate use of this approach is to be able to replace a wild-type gene with a mutant copy to test the function of the mutated gene.

PCR-based site-specific mutagenesis Remember that the amplification of a region of DNA by PCR involves a template molecule, which is often a gene or cDNA copy of a gene, and a pair of primers used to initiate DNA replication of the template. If the sequence of one of the primers is made to be slightly different than the template sequence, then as DNA amplification occurs most of the DNA molecules made in the PCR will end up with a specific mutation at a specific site. In this way different mutations can be introduced into a gene or cDNA and the effects of those mutations can then be analyzed in a variety of ways, some of which we explore in this section.

PCR-based random mutagenesis As we previously discussed, one of the polymerases used in PCR is called Taq polymerase. Unlike the polymerases used to replicate DNA in cells, Taq polymerase is not able to correct errors it makes during DNA synthesis. Taq polymerase will randomly introduce a mutation approximately every 45,000 nucleotides synthesized. Changing the chemical conditions of the PCR can further increase the mutation rate so that by performing repeated rounds of PCR, randomly mutagenized cloned genes or cDNA can be obtained. The effects of these mutations on gene product function can then be analyzed in a variety of ways. 350 part III Genetic and Molecular Biology

RNA interference can reduce the level of a gene product Although the ability to make and analyze mutants in vitro is ­extremely powerful, in many experimental systems it is not possible to introduce these constructs back into a living cell to assess their affects on phenotype. The technique of RNA interference (RNAi) allows researchers to reduce the amount of a gene product in living cells/organisms. This is the equivalent of a mutation that eliminates the function of a gene product. RNA interference acts to degrade or block translation of a specific mRNA in cells, which in turn reduces the level of the encoded protein. This technique takes advantage of systems in cells that have evolved to use small RNAs to control the level of gene expression posttranscriptionally (see chapter 16 for details). Using RNAi to reduce or eliminate the production of a specific protein requires the production of a short double-stranded RNA complementary to the mRNA that encodes the protein. A variety of techniques exist to create double-stranded RNA and introduce it into cells or organisms. RNAi then depends on the ­cellular mechanisms described in chapter 16. For any organism for which we have a complete genome sequence, a library of short double-stranded RNA molecules can be created to target every gene in the genome. These libraries can be used to systematically reduce expression of individual proteins or combinations of proteins so their roles in cells and organisms can be investigated.

The CRISPR/Cas9 system allows direct editing of the genome PCR-based mutagenesis allows the in vitro alteration of gene sequences and RNAi allows the reduction of gene products. These both provide indirect ways to investigate gene function in living cells. The next step is to directly edit genes in living cells. For research, this allows a direct assessment of how changing a protein affects its function, and for the clinic, it offers the possibility to alter genetic variants that cause genetic diseases. While this was once the province of science fiction, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system makes this not only possible, but relatively straightforward. Much as the study of host restriction in bacteria led to the discovery of restriction endonucleases, the study of how some ­bacteria could become “immune” to infection by a virus led to the discovery of the CRISPR/Cas9 system. Some bacteria appear to have a kind of adaptive immunity that involves a complex system that has evolved to incorporate part of the viral genome into the bacteria’s genome. This viral DNA can then be used to target future infection by a virus with the same DNA sequence. A bacterium uses the viral DNA to transcribe an RNA that is used as a “guide” by a nuclease (Cas9) to target DNA with the same sequence as the guide RNA. Once inside a cell, a guide RNA and the Cas9 protein associate, and can bind to a gene with sequences that match the guide RNA sequence (figure 17.9a). The Cas9 nuclease will cut the DNA where the guide RNA binds. Inaccurate repair of the cut

Cas9

CRISPR RNA (crRNA)

Cleavage Target Cleavage

Nucleasedeficient Cas9 (dCas9)

Activator

dsDNA

mRNA Activation

PAM

Repressor

Figure 17.9 The CRISPR/Cas9 genome editing system. a. A sequence-specific guide

RNA targets Cas9 to a target sequence where Cas9 cuts the target sequence. Repair can be inaccurate, resulting in gene inactivation, or accurate in the presence of a donor DNA sequence that results in replacement of the DNA target sequence with the donor sequence. b. Recombinant DNA technology can be used to target Cas9 to a gene and turn it on (activation), turn it off (inactivation), or reveal its location in the genome.  Source: New England BioLabs Inc., www.neb.com/tools-and-resources/feature-articles /crispr-cas9-and-targeted-genome-editing-a-new-era-in -molecular-biology

Repression

Donor DNA

Green fluorescent protein (GFP)

Insertion/ deletion New DNA

New DNA

Nonhomologous end joining (NHEJ)

Homology-directed repair (HDR)

a.

Visualization

b.

DNA can result in small deletions or insertions to the genomic sequence that can result in loss of gene function. Alternatively, if linear, double-stranded DNA is available, homology-directed repair can accurately repair the cut. This allows both restoring mutant alleles to wild type, and creating mutations in genes of interest. The technology continues to be improved, with Cas9 proteins isolated from different bacteria that are smaller and thus easier to transfer. Cas9 proteins are also being engineered to make the system more flexible and efficient. This technique has been used to inactivate the genome of HIV integrated into T cells grown in vitro, and has potential to treat genetic diseases. CRISPR/Cas9 can also be used to regulate gene ­expression. By constructing fusions between a Cas9 that lacks nuclease activity, and a transcriptional activator or repressor, genes can be selectively turned on or off. It is also possible to create fusions between fluorescent proteins and nuclease-­ deficient Cas9 proteins (figure 17.9b). This could simplify the detection and analysis of oncogenes that are amplified in certain cancers.

Learning Outcomes Review 17.3

Individuals can be identified using DNA fingerprinting. A specific set of STR loci makes up the CODIS database. PCR can be used to create site-specific or random mutations in DNA. RNA interference can be used to reduce levels of gene products. Genome-editing techniques can be used to remove gene function and to introduce new alleles in the chromosome. ■■

How does genome editing differ from creating specific mutations in vitro?

17.4 Constructing

and Using Transgenic Organisms

Learning Outcomes 1. Explain how the universal nature of the genetic code allows transgenesis. 2. Compare and contrast knockout, knockin, and conditional knockout mice. 3. Describe how transgenic plants are created.

In this section, we will explore how some of the technologies we have already considered can be used to construct transgenic organisms. These are organisms modified by addition of one or more genes from a different species. Any alteration of a genome through techniques other than conventional breeding produces an organism sometimes referred to as a genetically modified organism (GMO). First we will consider how transgenics are used for functional analysis of individual genes. In the remainder of the chapter we will consider applications in medicine, environmental biology, and agriculture.

The universal nature of the genetic code allows transgenesis A human gene can be placed into a mouse genome and the cells of the mouse will express the gene. Similarly, a human gene can be placed into the genome of an E. coli bacterium and the bacteria will make the encoded protein. This is because the cells of organisms of different species interpret genetic information identically. The genetic code chapter

17 Biotechnology 351

used to decipher the information in a gene is the same code used in bacteria, archaea, and eukaryotes. For example, the amino acid phenylalanine will always be coded for by the codon UUU; this is true whether we look at the genetic code in humans or examine the genetic code in E. coli. There are minor variations to the genetic code, however. For example, the genetic code of mitochondria is slightly different from the genetic code used to interpret nuclear genes.

Removal or addition of genes can reveal function The techniques for making recombinant DNA can be combined with other techniques to create organisms that have genes removed or have genes added. In some cases, a gene can be removed and then replaced with a version that has been genetically altered. The study of these animals and plants allows scientists to better understand the relationships between genotype and phenotype. By introducing mutant alleles into organisms, the effects of specific mutations in disease processes can be studied in the whole organism.

“Knockout” mice A “knockout” animal has had a gene inactivated so that the function of the gene is lost. It is possible to inactivate genes in a number of different experimental organisms. The most relevant to human

neo

genetics is the mouse, which has a rich genetic history. The effect of knocking out a gene can be assessed in the adult mouse—or if the gene is essential for survival, the developmental stage requiring the gene’s function can be identified. A streamlined description of the steps in creating a knockout mouse are outlined as follows and illustrated in figure 17.10: 1. The cloned gene is disrupted by replacing part of it with a marker gene using recombinant DNA techniques. The marker gene codes for resistance to the antibiotic neomycin in bacteria, which allows mouse cells to survive when grown in a medium containing the related drug G418. The construction is done such that the marker gene is flanked by the DNA normally flanking the gene of interest in the chromosome. 2. The interrupted gene is introduced into embryonic stem cells (ES cells). These cells are derived from early embryos and can develop into different adult tissues. In these cells, the gene can recombine with the chromosomal copy of the gene based on the flanking DNA. This is the same kind of recombination used to map genes (see chapter 13). The knockout gene with the drug resistance gene does not have an origin of replication, and thus it will be lost if no recombination occurs. Cells are grown in medium containing G418 to select for recombination

Figure 17.10 Construction of a knockout mouse. Steps

neo

in the construction of a knockout mouse. Some technical details have been omitted, but the basic concept is shown.

Gene to be knocked out

neo 1. Using recombinant DNA techniques, the gene encoding resistance to neomycin (neo) is inserted into the gene of interest, disrupting it. The neo gene also confers resistance to the drug G418, which kills mouse cells. This construct is then introduced into ES cells.

2. In some ES cells, the construct will recombine with the chromosomal copy of the gene to be knocked out. This replaces the chromosomal copy with the neo disrupted construct. This is the equivalent to a double crossover event in a genetic cross.

Embryonic stem (ES) cells with knocked-out gene

ES cells containing neo G418-containing medium

Dead cells without knocked-out gene

3. The ES cells are placed on G418containing medium. The G418 selects cells that have had a replacement event, and now contain a copy of the knockedout gene.

352 part III Genetic and Molecular Biology

Surrogate mouse Blastocyst

4. The ES cells containing the knocked-out gene are injected into a blastocyst stage embryo and then implanted into a female to complete development.

Heterozygous mouse carrying the knockout gene

Homozygous mouse for the knockout gene

5. Offspring will contain one chromosome with the gene of interest knocked out. Genetic crosses can then produce mice homozygous for the knocked-out gene to assess the phenotype. This can range from lethality to no visible effect depending on the gene.

events. (Only those containing the marker gene can grow in the presence of G418.) 3. ES cells containing the knocked-out allele are injected into a mouse embryo early in its development, which is then implanted into a pseudopregnant female (a female that has been mated with a vasectomized male and as a result has a receptive uterus). Some of the cells in the pups born to this female have one of the two alleles for the gene of interest knocked out and so these animals are chimeras. A chimeric mouse is mated with a normal mouse and some of the offspring will be completely heterozygous. In some cases there may be a phenotype that can be detected and analyzed if heterozygosity affects any biological function. If the mutation is recessive and not lethal, heterozygotes can then be crossed to generate homozygous mice. The homozygous mice can be analyzed for phenotypes.

“Knockin” mice Knockin mice have a normal allele replaced with an allele that has a specific genetic alteration. The ability to eliminate alleles provides insight into a complete loss of gene function, a so-called null allele, but does not allow analysis of alleles that alter but do not completely remove function. Specific mutations can be constructed in vitro, then introduced into the mouse to assess the effects of the alterations. This allows an investigator to use naturally occurring alleles, targeted mutations based on other information, or

literally any alteration desired. Mutations can be introduced that result in complete loss of function, partial loss of function, or even gain of function of the gene product.

Conditional inactivation allows the study of essential genes There are two major shortcomings to knocking out genes in mice. First, a gene knockout results in all cells of all tissues being affected by the inactivation. This is problematic for genes whose products affect more than one tissue. The phenotypic analysis of a knockout that affects multiple tissues can be complex and may not even be possible. The second problem has to do with developmentally important genes. A gene that is essential for early development will lead to embryonic lethality, and may leave nothing to analyze. ­Because of these limitations, ways to remove genes from the g­ enome in specific tissues and at specific developmental times were ­developed. Conditional inactivation allows a gene to be knocked out in cells of specific tissues or at a specific time in development. Conditionally knocking out a gene commonly uses the Cre-Lox recombination system from the bacterial virus called P1. Cre is an enzyme that catalyzes recombination between short DNA sequences called Lox sequences. If a gene of interest is flanked by Lox sites, then in the presence of the Cre enzyme, recombination between the Lox sites will remove the intervening sequence of DNA (the gene of interest) as a circular DNA that will be degraded (figure 17.11).

Cre Mouse

LoxP (Floxed) Mouse

Inducible or tissue-specific promoter

Stop

cre

Stop loxP

Target gene

Stop loxP

Selectable marker

Cre-LoxP Mouse Cells with active Cre recombinase Stop

cre loxP

Cells lacking active Cre recombinase

Selectable marker

Stop loxP

Target gene

Stop loxP

Selectable marker

loxP Target gene

Target gene function disrupted

Target gene function intact

Figure 17.11 Creating conditional and tissue-specific knockout mice. A Cre mouse expressing the Cre recombinase from an inducible or tissue-specific promoter is crossed with a mouse containing a “floxed” target gene. The “floxed” mouse is created using the same techniques for making a knockin mouse. Progeny will contain both the Cre-expressing gene and the “floxed” target gene. The promoter expressing Cre is turned on in the presence of an inducing molecule, or in a particular tissue. chapter

17 Biotechnology 353

Tissue-specific gene knockouts To perform a conditional inactivation using Cre-Lox, two lines of transgenic mice are constructed. The first line expresses the Cre enzyme from a promoter that is active only in the tissue of interest. In these mice, Cre will be produced only in the tissue of interest. The second line is a knockin mouse that contains the gene of interest flanked by Lox sequences. A sequence of DNA flanked by Lox sites is said to be “floxed.” Once the two lines are created, genetic crosses produce offspring in which all cells of the mouse contain the Cre recombinase and the allele to be knocked out. However, only in the tissues that express the Cre enzyme will there be recombination to remove the “floxed” DNA. This allows the effect of loss of function for the floxed gene in that tissue to be analyzed. Other tissues in the mouse will retain the normal allele and should not contribute to any phenotype associated with loss of the gene (figure 17.11).

Developmental knockouts To knock out a gene at a specific stage in development, a strategy is used similar to that for making a tissue-specific knockout. The only difference is that the Cre-expressing transgenic line will be engineered to express Cre at a specific developmental stage instead of in a specific tissue. An additional wrinkle on this strategy is to fuse Cre to a promoter that can be turned on when the mouse ingests a particular compound in its food or water. This allows analysis of developmental events after birth.

Plants require different techniques to be genetically modified Genetically modified plants are created using techniques that are considerably different from those employed to create genetically modified animals. Transgenic plants can be created by introducing

foreign DNA into plant cells by electroporation, physical bombardment, chemical treatment, and bacterial transfer. Whichever technique is used, it is not generally possible to specifically target a particular gene sequence, and integration of transgenes into plant genomes is random. Although this allows the introduction of a transgene to be expressed in the plant, it is a problem for knocking out a specific gene. It is desirable to introduce transgenes into crop species that are capable of increasing resistance to disease, frost, herbicides, and drought; also, creating crop plants with modified nutritional profiles would be beneficial to populations where nutritional deficiency is a problem.

Transforming plants with a bacterial pathogen Transgenes can be transferred into the genomes of certain plant species from the plant pathogen Agrobacterium tumefaciens. This bacterium harbors a plasmid called the Ti (tumor-inducing) plasmid. The Ti plasmid contains genes that normally cause the formation of a plant tumor—called a gall—in tissues of infected plants. The plasmid also contains genetic sequences responsible for transferring part of the plasmid from the bacterium into plant cells. The Ti plasmid can be isolated from Agrobacterium, and the genes responsible for gall formation can be removed and replaced with a gene of interest. Recombinant Ti plasmids containing a gene of interest can be reintroduced into Agrobacterium that can then be used to infect cells isolated from plants. During the infection process the bacterium transfers part of its Ti plasmid, including the gene of interest, into the plant cell and that piece of DNA can become integrated into the plant cell’s genome (figure 17.12). This process is called transformation. Under the correct nutritional and growth-stimulating conditions, the infected plant cells can be induced to produce roots and shoots. Eventually a mature plant in which all cells contain the transgene is grown.

Gene of interest Plasmid

Agrobacterium Plant nucleus

1. Plasmid is removed and cut open with restriction endonuclease.

2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium.

3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell.

4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene.

Figure 17.12 Creating transgenic plants using Agrobacterium transformation. Steps in the generation of transgenic plants using

Agrobacterium tumefaciens transformation. 354 part III Genetic and Molecular Biology

Transformation via particle bombardment Agrobacterium cannot infect all plant species, so not all plant species can be transformed using the method just described. When transformation of plant cells by Agrobacterium is not possible, DNA can be introduced into plant cells using physical techniques. A common approach to physically introducing recombinant DNA into plant cells is to coat gold or tungsten nanoparticles with recombinant DNA and to fire the particles at fragments of plant tissue. The DNA is carried into cells on the tiny particles, and when in the cells, the DNA may integrate into the genome. The plant tissue can then be grown in vitro, induced to differentiate, and eventually grown into a mature plant.

Algae can be used to manufacture biofuels

Learning Outcomes Review 17.4

The near-universal nature of the genetic code allows genes to be moved between different species. Knockout mice have specific alleles removed from the genome. Knockin mice have specific alleles replaced with an altered copy. Conditionally knockedin alleles can be activated in specific tissues or at specific times during development. Plants can be transformed with Ti plasmid DNA from Agrobacterium or by nanoparticle bombardment. ■■

Under what circumstances would it be appropriate to make a conditional knockout mouse versus a normal knockout mouse, and when might you need to make a knockin mouse?

17.5 Environmental

increase the sustainability of existing resources. Many applications of biotechnology in the environmental sciences focus on the recycling of waste materials such that the waste of one process can be the input to another process. Additionally, there is currently much interest in the use of biological processes in generating renewable fuel sources. Environmental biotechnology is not a new field, as biological processes have been used to manage wastewater since the mid-19th century. Technical advances in chemistry, genomics, genetics, and physics have accelerated our potential to develop technologies needed to tackle human-made environmental problems such as the global energy and water availability crises.

Applications

Learning Outcomes 1. Describe the benefits of biofuel production from algae. 2. Describe the factors that limit microbial degradation of hydrocarbons in natural environments. 3. Explain the importance of microorganisms in wastewater treatment.

Environmental biotechnology is the use of biological processes to protect, repair, and reduce human impacts on the environment or to

Biofuels are fuels derived from biomass made from recently fixed carbon sources, as opposed to fuels derived from “ancient” biomass such as fossil fuels. Common sources of biomass for biofuel generation include crop plants and algae. There are two common forms of liquid biofuel: ethanol derived from the fermentation of plant carbohydrates and biodiesel produced from the chemical transformation of plant or algal lipids. There are a number of species of algae that can be used to produce biodiesel; however, all are microalgae with cell diameters in the range of 3 to 30 µm. Biofuels produced from microalgae have several advantages over petroleum-based fossil fuels. They are renewable as long as carbon dioxide can be fixed into biomass by photosynthesis. They are more environmentally friendly than fossil fuels, given that carbon in the fuel is derived from atmospheric carbon dioxide, a greenhouse gas that contributes to climate change. The production of biodiesel from microalgae can also be combined with processes such as wastewater treatment or coupled to carbon dioxide capture systems of fossil fuel–burning industrial plants. Microalgae can be cultured in a variety of ways. The simplest culture system involves the creation of large open ponds. Although relatively cheap to construct and maintain, these ponds suffer from contamination by environmental microorganisms or algal predators. Also, they require large areas of land and cannot sustain very dense algal cultures. More expensive, but more easily controlled, are large self-contained culture systems that supply a source of water with dissolved nutrients and are combined with a light harvesting system through which the algal culture circulates (figure 17.13). These

Sunlight Gas and water conditioning

Algae growth

Algae harvesting

Oil extraction

Algae oil

Refinery

Biofuel

Nutrients

Recycled water CO2

Processing of remaining biomass

Figure 17.13 Using microalgae to make biofuel. Microalgae grown under certain conditions accumulate up to 50% of their biomass as lipids. Growth can be in ponds or, as shown here, photobioreactors. In some cases, carbon dioxide can be fed into the photobioreactors from industrial plants. Lipids harvested from the algae are processed and refined into biofuel. chapter

17 Biotechnology 355

photobioreactors are more easily controlled environments and can be fed with wastewater. Carbon dioxide can be bubbled through the system to provide a constant source of carbon for fixation by photosynthesis. Photosynthesis produces reduced carbon that can be metabolically converted to acetyl-coA, which is the building block of fatty acids. Some species of microalgae can also use heterotrophic nutritional strategies. These species absorb sugars from the culture medium and use those sugars for the formation of biomass for growth, or for the production of lipids. Yet other species can use both heterotrophic and photoautotrophic nutritional strategies simultaneously and these species produce some of the highest concentrations of lipid in their cells. Under conditions of stress, the algal cells can be composed of 40 to 50% triacylglycerides. A variety of techniques can be used to harvest the lipids from the algae, and this is one of the most expensive parts of algal biodiesel production. Once the lipids have been isolated from the algae, chemical treatments separate the glycerol from the fatty acid chains in the triglyceride. Further refinement yields a biodiesel product.

?

Inquiry question How might you genetically engineer

microalgae so that they are more efficient at making the lipids used to create biofuels?

Microorganisms can degrade hydrocarbons in the environment Our dependence on hydrocarbon-derived fuels—be they petroleumor biofuel-based—has an inevitable and serious consequence: those fuels can enter ecosystems and cause environmental damage. Certain hydrocarbons are persistent in terrestrial, aquatic, and marine ecosystems for decades and can accumulate on and in organisms, leading to death. Hydrocarbons that persist in the environment can dissipate slowly by evaporation of volatile compounds, by dissolution and dispersion in watery environments, and by oxidation in the presence of light. Importantly, certain microorganisms are capable of metabolizing hydrocarbon pollutants. The degradation or metabolism of hydrocarbon pollutants by microorganisms is called bioremediation. The rate at which these microorganisms metabolize hydrocarbons is affected by a number of variables including temperature, physical and chemical properties of the pollutant, oxygen availability, pH, salinity, other microorganisms present, and perhaps most critically, nutrient availability. Lab studies have shown that nutritional supplementation of certain microorganisms leads to a significant increase in the rate at which hydrocarbon pollutants can be metabolized. The addition of certain nutrients and other chemical additives to a polluted environment can increase the rate of bioremediation of hydrocarbon pollutants. Unfortunately, the increases in decontamination seen in the environment are not as great as those in the lab. An interesting possibility to increase the rate of decontamination by microorganisms is genetic engineering to optimize the pathways involved in hydrocarbon uptake and catabolism. Bioremediation of hydrocarbons is an important part of a hydrocarbon contamination cleanup plan. Importantly, when metabolized by microorganisms, hydrocarbons end up being metabolized into biomass or catabolized into carbon dioxide, so no additional toxic compounds are released into the environment. 356 part III Genetic and Molecular Biology

Wastewater treatment relies on the action of microorganisms Another example of using microorganisms to decontaminate a natural resource is the treatment of industrial and commercial wastewater. Wastewater can consist of raw sewage containing human and other animal fecal material as well as industrial contaminants. Untreated release of this wastewater would wreak havoc on ecosystems and spread disease. Wastewater is treated in two, or three, stages called primary, secondary, and advanced treatment. Microorganisms play critical roles in the secondary and sometimes advanced treatment stages. Primary treatment involves physical and chemical techniques to remove large and small particulate material. Secondary treatment can involve digestion by anaerobic bacteria, aerobic bacteria, or both (figure 17.14).

Aerobic treatment of wastewater Material from a primary treatment plant is fed into large aerated tanks that are continuously agitated where oxidative breakdown of any remaining organic material occurs. The bacteria and other organisms in the aerobic tanks form aggregates called floc. Degradation of the remaining carbon-containing compounds in the wastewater occurs on this floc, which is later removed in settling tanks. Wastewater is also treated on beds of crushed rock that contain bacterial communities, called biofilms. These perform the same function as the floc in agitated tanks. Treated water from the aerobic digester can be released into the environment, or further processed to produce potable water. Advanced treatment processes are used to remove inorganic contaminants such as phosphorus. This can be done by chemical addition, or using bacterial species capable of metabolizing dissolved phosphorus. The metabolized phosphorus is accumulated into cytoplasmic granules in the bacteria, which can be harvested and disposed of.

Anaerobic treatment of wastewater effluent Anaerobic digestion of wastewater occurs in sealed reactors called sludge digesters. In the sludge digester, anaerobic bacteria catabolize polysaccharides, lipids, and proteins remaining after primary treatment into their constituent monomers. The resulting fatty acids, amino acids, and sugars are metabolized into products such as acetate, hydrogen gas, and carbon dioxide. Archaebacteria in the sludge digester can convert these molecules to methane, carbon dioxide, and water. The methane can be burned to heat or power the wastewater treatment plant. Some of the sludge produced by the digester is cycled back into the system to maintain the population of bacteria required for digestion.

Learning Outcomes Review 17.5

Environmental biotechnology uses biological processes to protect or repair the environment. Microalgae can be used to produce biofuel. Benefits include increased sustainability and the coupling of fuel production to wastewater treatment. Nutrient supplementation of contaminated environments can stimulate hydrocarbon breakdown. Wastewater treatment processes using microorganisms occur in several stages, of which aerobic and anaerobic digestion involve the use of microorganisms. ■■

Could genetic engineering improve wastewater treatment?

Input of water from primary treatment

Water from primary clarifier

Aeration basin

Mixers for aeration

Distribution boom

Aerobic bacterial growth on rocks

Organic matter processing by suspended biomass

Treated water release Sludge

a.

Water sludge to disposal

Activated sludge biomass return Methane outlet

Figure 17.14 Using microorganisms to treat wastewater. 

Digester cover

Methane

Release of treated water

b.

Release to treated water

a. Organic matter in wastewater can be digested by aerobic bacteria in mixing basins. b. Alternatively, wastewater can be trickled over crushed rocks, and bacteria on the surface of the rocks aerobically digest the organic material in the wastewater. c. Organic material can also be removed from wastewater through the actions of anaerobic microorganisms. This process produces methane.  Source: Willey, J. M.,

Sherwood, L., Woolverton, C. J., Prescott, L. M., & Willey, J. M. (2011). Microbiology. New York: McGraw Hill.

Mixing Complex substrates

Effluent outlet

Fermentation Acetate, CO2, H2 CH4 + CO2

Important proteins can be produced using recombinant DNA

Solids

c.

17.6 Medical

genetic conditions and infectious diseases, and given us ways to make novel medicines. The application of biotechnology to medicine is not a new endeavor; however, advances in genetics, genomics, and recombinant DNA technologies have accelerated the rate of discovery in the last 20 years.

Applications

Learning Outcomes 1. Describe how recombinant DNA has been used in medical treatments and diagnostics. 2. Compare and contrast FISH and gene chips technologies for diagnosing disease. 3. Describe how immunoassays can be used to diagnose viral infections.

The manipulation of biological systems has led to major advances in health care. Specifically, biotechnology has provided novel diagnostic tools, allowed the treatment of previously untreatable

Diseases that are due to the body’s inability to produce a specific protein can be treated by providing the missing protein. Traditionally, these are isolated from animal tissues, but recombinant DNA allows us to express proteins in bacteria and other systems. The first medical product to be produced via recombinant DNA was insulin, which is normally produced in the pancreas, then transported via the bloodstream. Insulin is one of the hormones involved in regulating glucose levels (see chapters 44 and 46), and low levels of insulin cause blood sugar to rise. High blood sugar is a symptom of type I diabetes, which can be managed with daily insulin injections. The production of insulin using recombinant DNA is instructive because it shows both the promise of this technology, and also the challenges. In human cells, insulin is expressed as a single mRNA that is translated into a polypeptide that is cleaved into two shorter polypeptides that are then linked by disulfide bonds (figure 17.15a). Using recombinant DNA, a cDNA derived from the coding region of the insulin gene can be expressed in E. coli, and used to produce recombinant protein. However, E. coli will not process this protein as in human cells. To get around this, two cDNAs, which chapter

17 Biotechnology 357

In Humans Promoter

Exon

Intron

In Bacterial Culture Exon

Intron

Exon AmpR

β-gal

Bacterial promoter

Bacterial promoter

Insulin A chain minus introns and other “extra” sequence

Transcription Exon

β-gal

Insulin B chain minus introns and other “extra” sequence

Exon Transform into E. coli

Translation 108 amino acids

Preproinsulin Culture cells

Posttranslational modification Cut

Purify β-gal-insulin fusion proteins

Disulfide bonds form

Cut

A chain

Purify A and B chains

B chain

Cut A chain

NH2

B chain

NH2 Disulfide bond

Disulfide bond

a.

COOH COOH

Active insulin

b.

Figure 17.15 Making insulin in genetically engineered E. coli. a. In human cells one preproinsulin polypeptide is processed posttranslationally into insulin chains A and B that associate via disulfide bonds to form mature insulin. b. Two cDNAs corresponding to the gene sequences for insulin chain A and chain B are cloned into plasmids and introduced into different E. coli. Cultures of the two types of E. coli express either chain A or chain B. Chains A and B are purified from the different E. coli and, when mixed, associate into active mature insulin.

encode the two polypeptides that make up active insulin protein, are used instead of a single full-length cDNA. Each of these cDNAs is spliced into a separate plasmid vector that can be expressed in E. coli. Proteins isolated from two strains of E. coli expressing the individual insulin polypeptides can then be purified and mixed together to make a functional insulin molecule (figure 17.15b). When purified of other contaminating bacterial proteins, the insulin reaches very high concentrations. Unfortunately, at these high concentrations, the insulin molecules stick together, impairing their function. To solve this last problem, specific codons in the insulin cDNA sequences were altered so that the resulting proteins don’t stick together. This allows the production of high concentrations of highly effective insulin.

Fluorescent in situ hybridization (FISH) can detect gross chromosomal rearrangements Fluorescent in situ hybridization (FISH) takes advantage of the ability of DNA to be reversibly denatured and renatured. Many techniques in molecular biology use hybridization to detect a specific DNA in a complex mixture. Hybridization uses a probe 358 part III Genetic and Molecular Biology

that is complementary to the sequence of interest, and either a fluorescent or a radioactive label. Both probe and target DNA are denatured and mixed together. When conditions are restored to allow renaturation, the probe will find any complementary sequences in the mixture and form a hybrid. In FISH, the target DNA is a metaphase spread of chromosomes, or cells with intact chromosomes. FISH allows the detection of gross chromosomal abnormalities, such as large deletions, inversions, duplications, and translocations (see chapter 15 for descriptions). Aneuploidy and chromosomal abnormalities are associated with many forms of cancer. About 25% of invasive breast cancers involve duplications of a gene called HER2. Instead of the usual two copies expected in a diploid cell, there can be hundreds of copies. The HER2 gene encodes a receptor protein that initiates a signal transduction pathway for cell proliferation. Therefore, extra copies of HER2 can lead to uncontrolled cell growth and tumor formation. Drugs that block cell signaling through the HER2 protein can be used to treat these kind of tumors. However, these drugs are only effective in treating tumors caused by HER2 duplications, making data on the copy number of HER2 genes vital for clinicians . These data allow treatment to be personalized based on a patient’s specific genetic profile.

Probe DNA 1 Fluorescent labeling of probe DNA Fluorescent signal 2 FISH probe attaching to DNA molecule

b.

3 Hybridization

a.

Figure 17.16 Fluorescent in situ hybridization. 

a. Fluorescent probes bind to specific DNA sequences in chromosomes. b. Only two spots of fluorescence are visible in the nuclei of the tumor cells, indicating that HER2 is not amplified. c. Multiple large c. fluorescent spots in the tumor cells indicate amplification of HER2.

FISH is used to detect HER2 in cells taken from a tumor biopsy. Tumor cells are collected and treated to expose the chromosomes. A fluorescent labeled HER2 probe is used for hybridization to identify HER2 DNA in the chromosomes (figure 17.16a). When cells are illuminated with UV light, which causes the probe to fluoresce, HER2 sequences are visible. In normal cells, or in cells from breast cancers not caused by duplication of the HER2 gene, just two small dots of fluorescence will be seen in the microscope (figure 17.16b). If the breast cancer cells contain more than two copies of the gene, then there will be multiple, much larger dots of fluorescence (figure 17.16c). This is an example of personalized medicine where drugs are administered only when necessary.

Data analysis If you performed a FISH analysis on

human cells in G2 of the cell cycle using a probe that recognized telomeric DNA (DNA on the tips of the chromosomes), how many spots would you expect to see in the microscope?

Gene chips can identify genetic markers for disease Another use of hybridization involves so-called gene chips, also known as DNA microarrays, which are collections of hundreds to thousands of different known DNA sequences immobilized on a solid surface. The DNA sequences can be chemically synthesized, or amplified by PCR or RT-PCR. By using different gene chips,

and different experimental conditions, a variety of kinds of questions can be assessed. A disease marker, or biomarker, is a biological molecule found in affected individuals, and not in unaffected individuals. Biomarkers can be diagnostic of a disease state, or of the progress of a disease, and can help monitor the response to therapy. If a specific gene is expressed only in affected individuals, then the mRNA encoded by that gene can be a biomarker. For example, individuals with some autoimmune diseases or chronic inflammatory conditions express different sets of genes in immune cells than healthy individuals express. Gene chips can be used to monitor gene expression, including the relative levels of expression for different genes. Gene chips are also used to genotype SNPs in an individual, with literally hundreds of thousands of SNPs on a single chip. This allows the GWAS studies discussed in chapter 13 to collect very large data sets quickly and cheaply. If you have your DNA analyzed by any of the companies that offer to provide you information on your ancestry, they are not sequencing your entire genome; they are using microarrays with thousands to millions of informative SNPs. Taken together, technologies such as FISH and gene chips are changing the way that medicine is administered. The more that can be learned about the specific underlying causes of a disease on an individual basis, the more specific and personalized the health care provided. These technologies face some challenges, however. First, they require great skill to be successfully used; second, they are relatively expensive; and third, not all diseases are caused or influenced by factors specific to the individual. chapter

17 Biotechnology 359

Sample molecule

Read plate at 450 nm Well coated with antibody specific to sample molecule

1. Incubate

1. Wash 2. Add detecting antibody 3. Incubate

1. Wash 2. Add substrate 3. Incubate

a.

Figure 17.17 Detecting virus using an immunoassay. 

Microtiter plate

Enzyme substrate

Conjugated enzyme

7 Color reaction product

5 HIV-1/2 protein antigen conjugate 1 HIV-1/2 protein antigen

6

4 HIV protein 3 HIV-1/2 specific antibody 2 HIV-1/2 capture antibody

b. Fourth-generation HIV antibody and p24 ELISA test (1) HIV proteins and (2) HIV capture antibodies are attached to wells; (3) patient sample is added to wells that may contain antibodies to virus which can bind the HIV protein in the well or (4) HIV proteins that will be bound by the HIV capture antibodies; (5) addition of HIV protein with an attached enzyme or (6) anti-HIV protein antibody with an attached enzyme is added to the well; finally, (7) a substrate acted upon by the enzyme is added that is converted to a colored product.

Immunoassays can rapidly diagnose viral infections Immunoassays are tests that allow the detection of a molecule through the use of an antibody. Unlike gene chips or FISH, immunoassays are relatively cheap and easy to use. Antibodies are protein molecules produced by most animals in response to infection by pathogens (see chapter 50). Antibodies very specifically recognize a pathogen and target it for destruction by the immune system. Biotechnology companies can make antibodies against a range of viruses and bacteria. These antibodies can be used in immunoassays to detect whether there is virus in a sample collected from a patient. An antibody against a specific virus can be coated into a plastic well on a microtiter plate (figure 17.17a). When an unknown sample is added to the well, if the virus is present, it will be bound by the antibody. Bound virus can be detected using a second antibody against the virus that has been linked to a fluorescent molecule, or an enzyme that produces a colored product from a suitable substrate. The amount of fluorescence or colored 360 part III Genetic and Molecular Biology

a. Steps in an immunoassay to detect a virus in a sample. b. Modern HIV immunoassays have both viral proteins and antibodies to the virus bound to the well of the plate. Virus and antibodies against the virus in a sample will bind to the plate and can be detected using another antibody that produces a colored substance.

product made by the enzyme is proportional to the amount of virus present. This same technology can be used to detect antibodies produced by your immune system in response to infection. A variation of this technology is used in the diagnosis of human immunodeficiency virus (HIV) infection (figure 17.17b). In HIV immunoassays, an antibody recognizing HIV and a specific protein from HIV are coated onto the well of a microtiter plate. When a sample from a patient is added to the well, the ­virus binds to the HIV antibody, and any antibodies that are ­present in the blood that have been produced in response to the infection will recognize and bind to the HIV protein in the well. After washing, a mix of antibodies recognizing HIV protein linked to an enzyme and a specific HIV protein linked to an enzyme are added to the well. After more washing, any enzymes linked to the antibodies or HIV proteins that remain bound produce an easily detectable color change.

There are a variety of new vaccine technologies Traditional vaccines for viral pathogens have used two main technologies: inactivated virus and weakened (or attenuated) virus. In the first case, the actual virus is used, but it is chemically inactivated such that it is no longer infectious. Weakened viruses can still replicate, but do not cause severe symptoms. These are made by passaging a virus through cells, accumulating mutations, until a less infectious virus can be isolated, or using recombinant DNA technologies to directly alter viral genetic material. New vaccine technologies fall into three categories: nucleic acid vaccines, viral-vector vaccines, and subunit (recombinant protein) vaccines. The common thread in all of these is that they use a variety of technologies to produce a single protein, or even part of a protein, to be used as an antigen to stimulate an immune response (see chapter 50). This requires significant knowledge about the infectious agent, and the immune response to it. For example, with the COVID-19 pandemic of 2020, while an enormous amount of research was devoted to the SARS-CoV-2 virus,

this effort was also aided by work on the original SARS virus. It should also be noted that very few of these, mainly subunit or viral vector vaccines, have been approved for use in humans. We will use vaccines against SARS-CoV-2 to illustrate how these technologies work. It was known from work on SARS that the antigen that stimulated the strongest immune response was the spike (S) protein found on the surface of the virus, which is also involved in binding to the ACE2 receptor protein on cells. The DNA and mRNA vaccines both encode this S protein and are intended to enter cells and cause the production of S protein, which will then stimulate the immune system. Subunit vaccines skip this step and consist of just the S protein, or the S protein attached to the surface of a nanoparticle. Lastly, the viral vector vaccines consist of other innocuous human viruses that contain the gene for the S protein. These are usually the same virus vectors that are used in gene therapy experiments. All of these vaccine types have been in development for a number of years, but very few have received regulatory approval for use in humans. By the end of 2020, there were almost 200 candidate vaccines based on these technologies being developed for SARS-CoV-2. In response to the COVID-19 pandemic, two mRNA vaccines were given emergency approval in the U.S., the first mRNA vaccines of any kind to receive approval for use in humans. Both vaccines appear to be very effective at preventing severe illness, although how long this immunity will persist is unknown. It is also not clear if these are so-called sterilizing vaccines that prevent infection, and thus break the chain of transmission completely.

Gene and stem cell therapy and gene editing are all moving into the clinic From the earliest days of “genetic engineering,” biologists envisioned using this technology to restore or replace defective genes in humans. The first clinical trial in the U.S. of gene therapy was in 1990 for a rare inherited form of severe immune deficiency. This trial used autologous (from the patient) T cells to which a normal copy of a defective gene had been added. The trial was a partial success, and since that first trial, more than 2200 clinical trials have occurred worldwide, with the majority (63%) in the U.S. Despite this, there have been only a few gene therapy products approved. While gene therapy targets genetic diseases, stem cell therapy seeks to treat degenerative diseases, or tissue damage from injury, by replacing damaged tissue. The most common use of stem cells has been bone marrow transplantation, which is now used for 50,000 patients annually worldwide. Bone marrow contains stem cells that produce the different types of blood cells, and transplantation is used to treat a number of blood disorders, including various types of leukemia and anemia. This technique has also been successful in stopping progression of multiple ­sclerosis in some patients because it results in a complete reprogramming of the immune system. The majority of approved stem cell therapies use mesenchymal stem cells. These stem cells can differentiate into cell types that form connective tissues such as bone and cartilage. These therapies can promote connective tissue formation and repair, and reduce inflammation. Stem cell therapies for the treatment of the tissue damage caused by heart attacks are also moving through

clinical trials. Despite some successes, stem cell therapies face significant hurdles before they can realize the potential promised by researchers. These two kinds of therapy can be extended by new genome editing technology (section 17.4). In the most far-reaching form of this therapy, induced pluripotent stem cells (see chapter 19) derived from a patient can have defective genes restored, before reintroduction back into the patient. This is already being considered for a number of genetic diseases that result in blindness. Genetic diseases that affect a specific tissue are all potential targets for this methodology. Work is ongoing for treatment of diabetes, and of blood disorders such as sickle cell anemia. This kind of technology can also be used to treat infectious disease by targeting cellular receptors used by infectious agents to gain entry into cells. In a 2014 clinical trial, researchers disrupted the CCR5 gene in patient-derived CD4+ T cells. Infusion of these cells showed some promise in lessening the effects of HIV infection (see chapter 26). There are now multiple clinical trials using CRISPR/Cas9 systems to edit the CCR5 gene in T cells. Because of ethical issues, including the anticipated high cost of such therapy, the most important use of this technology may be for research. Cells taken from patients suffering from specific diseases can be used to create stem cells that represent a kind of “disease-in-a-dish.” These cells can be used for research into disease mechanisms and into potential drug therapies.

Learning Outcomes Review 17.6

Recombinant DNA technology allows the mass production of human gene products. FISH is used to detect chromosomal rearrangements, including those involved in cancers. This diagnostic information allows personalization of treatment. Gene chips are used to determine patterns of gene expression for many genes, or to detect many SNPs, at a time. Immunoassays are the basis of rapid and cheap diagnostic kits for viral infections. New vaccine technologies are being used for viral infections. Gene therapy may allow treatment of genetic diseases, and stem cell therapy combined with gene editing may allow treatment of tissue damage due to chronic disease or injury. ■■

Explain how to use genetic engineering to produce a specific protein product.

17.7 Agricultural

Applications

Learning Outcomes 1. Describe the benefits of creating transgenic crops. 2. Identify the most common forms of genetically modified plants. 3. Evaluate issues on each side of the transgenic crop debate.

Possibly the greatest impact of biotechnology has been in its application to agriculture. Transgenic crops are being created that resist disease, are tolerant of herbicides and drought, and have chapter

17 Biotechnology 361

SCIENTIFIC THINKING Hypothesis: Petunias can acquire tolerance to the herbicide glyphosate by overexpressing EPSP synthase. Prediction: Transgenic petunia plants with a chimeric EPSP synthase gene with strong promoter will be glyphosate tolerant. Test: 1. Use restriction enzymes and ligase to “paste” the cauliflower mosaic virus promoter (35S) to the EPSP synthase gene and insert the construct in Ti plasmids. 2. Transform Agrobacterium with the recombinant plasmid. 3. Infect petunia cells and regenerate plants. Regenerate uninfected plants as controls. 4. Challenge plants with glyphosate.

35S

EPSP synthase

Agrobacterium

Glyphosate

Ti plasmid

Cultured petunia cells

Transformed, regenerated petunia plant

Nontolerant petunia

Tolerant petunia

Result: Glyphosate kills control plants, but not transgenic plants. Conclusion: Additional EPSP synthase provides glyphosate tolerance. Further Experiments: The transgenic plants are tolerant, but not resistant (note bleaching at shoot tip). How could you determine if additional copies of the gene would increase tolerance? Can you think of any downsides to expressing too much EPSP synthase in petunia?

Figure 17.18 Genetically engineered herbicide resistance.

improved nutritional quality. Plants are also being used to produce pharmaceuticals, and domesticated animals are being genetically modified to produce biologically active compounds.

Herbicide-resistant crops allow for no-till planting Tilling is the process in which soils are turned over to prepare them for planting. This acts to remove weeds, which compete with crop plants for nutrients, and improve irrigation. Soils that are not tilled have greater levels of organic and inorganic nutrients that promote growth, often retain more water, and are less prone to soil erosion. There is an economic cost to tilling because it requires expensive equipment and takes time to perform. Weeds can also be removed from crop fields by the application of herbicides, but most ­common herbicides are not particularly discriminating and will kill a variety of different plant species. If crops are planted without the need for tilling, farms can be more efficient—which can translate into economic gains. Agriculturally important broadleaf plants such as soybeans have been genetically engineered to be resistant to glyphosate, a powerful, biodegradable herbicide that kills most actively growing plants (figure 17.18). Glyphosate works by inhibiting an enzyme called 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which plants require to make aromatic amino acids. Animals do not make aromatic amino acids; rather, they are ­obtained from the diet, so ­animals living in and around crops, or in waterways receiving crop 362 part III Genetic and Molecular Biology

runoff, are unaffected by g­ lyphosate. To make glyphosate-resistant plants, scientists used a Ti plasmid to insert extra copies of the EPSP synthase gene into plants. These engineered plants produce 20 times the normal level of EPSP synthase, enabling them to synthesize aromatic amino acids and grow despite glyphosate’s inhibition of the enzyme (figure 17.18). These advances are of great interest to farmers because a crop resistant to glyphosate does not have to be weeded through tilling. Because glyphosate is a broad-spectrum herbicide, farmers don’t need to employ a variety of different herbicides, most of which kill only a few kinds of weeds. Furthermore, unlike many other herbicides, glyphosate breaks down readily in the environment. Six important crop plants have been modified to be glyphosateresistant: maize (corn), cotton, soybeans, canola, sugar beet, and alfalfa. The use of glyphosate-resistant soy has been especially popular, accounting for 60% of the global area of transgenic crops grown worldwide. In the United States, 90% of soy currently grown is transgenic. Other commercially important crops, such as barley, rice, and wheat, have been made resistant to a range of other herbicides.

Bt crops are resistant to some insect pests Many commercially important plants are sensitive to insect pests, and the usual defense against this has been insecticides. Over 40% of the chemical insecticides used today are targeted against boll

Daffodil phytoene synthase gene (psy)

Bacterial carotene desaturase gene (crtI )

Figure 17.19 Construction of Golden Rice. Three daffodil

Daffodil lycopene β-cyclase gene (lcy)

genes were added to the rice genome to allow synthesis of β-carotene in endosperm. The source of the genes and the pathway for synthesis of β-carotene is shown. The result is Golden Rice, which contains enriched levels of β-carotene in endosperm.

Genes introduced into rice genome Rice chromosome

psy

crtI

lcy

Phytoene synthase

Carotene desaturase

β-Cyclase

Expression in endosperm

geranylgeranyl diphosphate

Phytoene

Lycopene

β-Carotene (Provitamin A)

weevils, bollworms, and other insects that eat ­ cotton plants. Scientists have produced plants that are resistant to insect pests, removing the need to use many externally applied insecticides. The approach is to insert into crop plants genes encoding proteins harmful to the pests, but harmless to other organisms. The most commonly used protein is a toxin produced by the soil bacterium Bacillus thuringiensis (Bt toxin). When insects ingest Bt toxin, endogenous enzymes convert it into an insect-specific toxin, causing paralysis and death. Because these enzymes are not found in other animals, the protein is harmless to them. Many of the same crops that have been modified for herbicide resistance have also been modified for insect resistance using the Bt toxin. The use of Bt maize is the second most common genetically modified (GM) crop, representing 14% of global area of GM crops in nine countries. The global distribution of these crops is also similar to that of the herbicide-resistant relatives. Given the popularity of both of these types of crop modifications, it is not surprising that they have also been combined, socalled stacked GM crops, in both maize and cotton. Stacked crops now represent 9% of global area of GM crops.

into rice to complete the biosynthetic pathway producing β-carotene in endosperm (figure 17.19). This case of genetic engineering is interesting for two ­reasons. First, it introduces a new biochemical pathway in tissue of the transgenic plants. Second, expression of β-carotene in the endosperm could not have been achieved by conventional breeding, as no known rice cultivar produces these enzymes in endosperm. A second-generation rice cultivar that makes much higher levels of β-carotene has also been produced by using the gene for phytoene synthase from maize in place of the original daffodil gene. Golden Rice was originally constructed in a public facility in Switzerland and made available for free with no commercial incentives. Since its creation, Golden Rice has been improved by public groups and by industry scientists, and these improved versions are also being made available without commercial strings attached.

Golden Rice shows the potential of transgenic crops

The adoption of transgenic crops has been resisted in some places for a variety of reasons. Some people have wondered about the safety of these crops for human consumption, the likelihood of introduced genes moving into wild relatives, and the possible loss of biodiversity associated with these crops. On the side promoting the use of transgenic crops are the multinational companies using these technologies to produce seeds for the various transgenic crops. Also on this side are groups n­ oting the benefits of transgenic crops like Golden Rice in feeding the developing world and preventing nutritional deficiencies. On the opposing side are a variety of political and social organizations that raise concerns for the ecological and health implications of genetically modified foods. Scientists can be found on both sides of the debate. The controversy originally centered on the safety of consuming foods containing introduced genes. In the United States, this issue has been “settled” for the crops already mentioned, and a large amount of transgenic soy and maize is consumed in this country. There remains a perceived risk of allergic reactions and

Golden Rice is a transgenic cultivar of rice, genetically modified to express β-carotene, the precursor of vitamin A. Ingested β-carotene can be converted to vitamin A in the body to alleviate the symptoms of vitamin A deficiency. The World Health Organization (WHO) estimates that vitamin A deficiency affects between 140 and 250 million preschool children worldwide, 250,000 to 500,000 of whom become blind. The deficiency is especially severe in developing countries where the major food is rice. Golden Rice is named for its distinctive color imparted by the presence of β-carotene in the endosperm (the outer layer of rice that has been milled). Rice does not normally make β-carotene in endosperm tissue, but does produce a precursor, geranylgeranyl diphosphate (GGPP), that can be converted by three enzymes— phytoene synthase, phytoene desaturase, and lycopene β-cyclase— to β-carotene. These three genes were altered using recombinant DNA technologies to be expressed in endosperm and introduced

Transgenic crops raise a number of social issues

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17 Biotechnology 363

longer-term effects, although there is no documentation of adverse effects on human health. Existing crops will be monitored for ­adverse effects, and each new modification will require regulatory approval for human consumption. Also concerning is the potential for spread of the transgenes from a transgenic crop to a non-transgenic crop. The amount of crosspollination between plants of the same species varies. Soybeans tend to self-pollinate with limited outcrossing. Corn, however, freely outcrosses and so genes from transgenic plants move more frequently to nontransgenic corn plants. This is a concern for organic farmers with fields close to transgenic corn fields because current regulations for organic certification exclude transgenic crops. The amount of outcrossing depends on the proximity of crops, wind speed, and wind direction, as well as temperature and humidity. For plants that depend on pollinators for reproduction, the range of species the pollinator ­visits and the distance it travels are also factors. Gene flow can also occur between related species through hybridization. In the United States, at least 15 weedy species have hybridized with crop plants. For two of the major U.S. crops, corn and soybean, no closely related weedy species reproduce with them, so this is an unlikely mechanism for acquiring herbicide resistance. Sunflowers, however, were first domesticated in the United States, and the risk of a transgene moving into a weedy

population is a concern. One study revealed that 10 to 33% of wild sunflowers had hybridized with domesticated varieties. In the case of herbicide-resistant transgenic crop plants, the repeated application of a single herbicide over a number of years creates selective pressure on weedy species. Over time, weeds with resistance to the herbicide will increase in frequency, and the herbicide becomes ineffective. Almost 200 weed species worldwide have acquired resistance to one or more herbicides. Management practices that reduce herbicide use and vary the herbicides used can slow the appearance of herbicide resistance.

Learning Outcomes Review 17.7

To date, herbicide resistance, pathogen protection, nutritional enhancement, and drug production have been targets of agricultural genetic engineering. Controversy regarding the use of transgenic plants has centered on the potential of unforeseen effects on human health and on the environment. ■■

Grasses resistant to glyphosate have been constructed, but never released as a product. What might keep a grass seed company from releasing this?

Chapter Review Professor Stanley N. Cohen/Science Source

17.1 Recombinant DNA

Restriction endonucleases cleave DNA at specific sites. Different DNA fragments generated by cleavage with the same type II restriction enzyme can be recombined (figure 17.1). Gel electrophoresis separates DNA fragments. DNA fragments can be separated based on size through a gel matrix driven by an electric field (figure 17.2). DNA fragments are combined to make recombinant molecules. A recombinant DNA molecule consists of a hybrid made from DNA. Recombinant DNA molecules are made by joining DNA fragments from different sources using DNA ligase (figure 17.3). Reverse transcriptase makes DNA from RNA. Reverse transcriptase uses RNA as a template to make a DNA molecule. The DNA is called cDNA (figure 17.4). DNA libraries are collections of recombinant DNA molecules. DNA libraries hold pieces of DNA and can be stored and replicated in a host organism (figure 17.5).

364 part III Genetic and Molecular Biology

17.2 Amplifying DNA using the Polymerase

Chain Reaction

PCR mimics DNA replication. PCR amplifies small DNA fragments using short primers that flank the region to be amplified. PCR uses cycles of heating, cooling, and synthesis with a thermostable polymerase (figure 17.6). Reverse transcription PCR makes amplified DNA from mRNA. Reverse transcriptase can be used to make cDNA. Combined with PCR, this can amplify sequences that encode proteins. Quantitative RT-PCR can determine levels of an mRNA. RT-PCR using fluorescent DNA binding dyes or labeled primers allows quantitation of a specific mRNA (figure 17.7) PCR is the basis of many sequencing technologies. Many DNA sequencing technologies rely on variations of PCR to generate starting material. These have greatly lowered costs. PCR is used in diagnostic tests for many diseases. PCR is the basis for very sensitive test kits for any pathogen that has nucleic acid.

17.3 Creating, Correcting, and Analyzing

Genetic Variation

Forensics uses DNA fingerprinting to identify individuals. DNA fingerprinting uses short tandem repeats (STRs) that vary in number between individuals. The CODIS database stores data on 13 different STR loci (figure 17.8). PCR can be used to create point mutations in specific genes. Altering a nucleotide in a PCR primer can be used to create site-specific mutations. The higher error rate of some DNA polymerases used in PCR can be used to create random mutations. RNA interference can reduce the level of a gene product. Short double-stranded RNAs introduced into cells will reduce the expression of specific genes. Libraries of these interfering RNAs can be analyzed to identify specific proteins involved in a biological process. The CRISPR/Cas9 system allows direct editing of the genome. Genes can be edited in vivo using the CRISPR/Cas9 system. This system introduces specific cuts to DNA and allows for either error-prone repair to create deletions or DNA-directed repair to change alleles. (figure 17.9).

17.4 Constructing and Using Transgenic Organisms The universal nature of the genetic code allows transgenesis. The universality of the genetic code means that genes from one organism can be expressed in other organisms. Removal or addition of genes can reveal function. In “knockout” mice, a gene is inactivated to study its function (figure 17.10). In “knockin” mice, a gene is replaced with a variant to assess the effects of specific genetic changes. Conditional inactivation allows the study of essential genes. Removing gene function from a specific tissue or at a specific developmental time point allows essential genes to be studied. One technology used is the Cre-Lox system (figure 17.11). Plants require different techniques to be genetically modified. Transgenic plants can be created by transformation with Agrobacterium and a recombinant Ti plasmid or particle bombardment (figure 17.12).

17.5 Environmental Applications Algae can be used to manufacture biofuels. Lipids harvested from microalgae can be converted into biodiesel. Some microalgae produce up to 50% of their biomass in lipids (figure 17.13). Microorganisms can degrade hydrocarbons in the environment. Nutritional supplementation of different environments can stimulate some bacteria to degrade hydrocarbons to remove toxic oil products.

Wastewater treatment relies on the action of microorganisms. The bulk of organic matter in wastewater is removed by anaerobic and/or aerobic bacteria. Further treatments can remove some inorganic contaminants (figure 17.14).

17.6 Medical Applications Important proteins can be produced using recombinant DNA. Recombinant DNA can be used to express human proteins in bacteria and yeast. Insulin to treat diabetes was the first such product (figure 17.15). Fluorescent in situ hybridization (FISH) can detect gross chromosomal rearrangements. FISH uses fluorescently labeled DNA probes that hybridize to complementary sequences in chromosomes. This can identify chromosomal alterations involved in disease such as amplification of HER2 gene, which can help design personalized treatment (figure 17.16). Gene chips can identify genetic markers for disease. Gene chips are used to analyze patterns of gene expression in diseased and healthy tissue. This can aid in design of clinical interventions. Immunoassays can rapidly diagnose viral infections. Antibodies can be used to detect specific molecules in clinical or ex­­perimental samples. Antibodies on a solid surface bind specific molecules, which are detected by a second antibody linked to an enzyme (figure 17.17). There are a variety of new vaccine technologies. New vaccines are being designed using nucleic acid, viral subunits, and modified innocuous viruses. This research has been stimulated by the COVID pandemic. Gene and stem cell therapy and gene editing are all moving into the clinic. Gene therapy may allow treatment of genetic diseases, and stem cell therapy combined with gene editing may allow treatment of tissue damage due to chronic disease or injury.

17.7 Agricultural Applications Herbicide-resistant crops allow for no-till planting. Herbicide-resistant crops reduce both the need for tilling and the associated fossil fuel consumption (figure 17.18). Bt crops are resistant to some insect pests. The development of insect-resistant Bt plants reduces the use of insecticides. Golden Rice shows the potential of transgenic crops. Golden Rice produces β-carotene, the precursor for vitamin A (figure 17.19). This may be used to reduce blindness from malnutrition. Transgenic crops raise a number of social issues. There are concerns about allergic reactions to transgenic plants, but none have been documented. The spread of transgenes into noncultivated plants is being followed. There are concerns about resistance to herbicides from overuse.

chapter

17 Biotechnology 365

Visual Summary DNA technology

Biotechnology

includes

Applications

includes

involves Recombinant DNA for

types

Molecular cloning techniques used Restriction endonucleases Gel electrophoresis

cDNA

DNA libraries

include

created and analyzed by

PCR and RT-PCR

DNA fingerprinting

used for

in vitro mutagenesis

Quantifying DNA or RNA

Transgenic animals

DNA sequencing

Transgenic plants

Knockout mice essential genes

Genome editing

and

include

remove genes

RNA interference

and

Environmental applications

Transgenic organisms

Genetic variation

Polymerase chain reaction

add genes

Diagnostic kits

Medical applications

include

Agricultural applications include

Protein drugs

Herbicide resistance

Hydrocarbon removal

FISH

Insect resistance

Wastewater treatment

Gene chips

Biofuels

Immunoassays Conditional inactivation

Knockin mice

Vaccines Gene therapy

Review Questions Professor Stanley N. Cohen/Science Source

U N D E R S TA N D 1. You study a gene known to be important in the early stages of heart development. Loss of the gene is also suspected to play a role in triggering lung cancer. What kind of transgenic mouse would you use to study your gene in vivo? a. b. c. d.

Knockout Knockin Conditional knockout All of the choices are correct.

The positive charge on DNA The size of the DNA fragments The sequence of the fragments The presence of a dye

About 100 About 1000

366 part III Genetic and Molecular Biology

c. d.

About 10,000 About 1010

It is likely to contain high levels of the HER2 mRNA. It is likely to contain high levels of the HER2 protein. The HER2 gene is amplified, and treatments targeting the HER2 protein may not be suitable. The HER2 gene is not amplified, and so particular treatments targeting the HER2 protein may not be suitable.

5. In terms of studying gene function, what is the main benefit that genome editing has over RNAi? a. b.

3. If a PCR is started using 10 pieces of template DNA, how many pieces of DNA would there be after 10 cycles? a. b.

a. b. c. d.

2. What is the basis of separation of different DNA fragments by gel electrophoresis? a. b. c. d.

4. FISH analysis of a breast tumor biopsy for HER2 gene reveals two spots of fluorescence in cells. What conclusion do these data support about the tumor?

c. d.

Genome editing can eliminate gene function; RNAi simply reduces the levels of gene products. Genome editing does not require that recombinant DNA be produced, whereas RNAi always does. Genome editing always works, but RNAi rarely works. Genome editing is done in bacteria, which are easier to manipulate than the eukaryotic cells in which RNAi is done.

6. The Ti plasmid of Agrobacterium usually induces tumors called galls on infected plants. When Agrobacterium is used to make transgenic plants, why does no gall form? a. b. c. d.

The introduced transgene blocks the action of the gallinducing genes. The gall-inducing genes are removed and replaced with the transgene. The Ti plasmid is removed from Agrobacterium used to make transgenic plants. The Ti plasmid is mutated by PCR so that it cannot replicate in infected plants.

A P P LY

Sample A Sample B

c. d.

Sample C Sample D

2. Which of the following statements is accurate for DNA replication in your cells, but not PCR? a. b. c. d.

a. b. c. d.

The genetic code of bacteria is significantly different from the genetic code of humans. The bacterial cell will be unable to posttranslationally process the insulin peptide sequence. There is no way to get the bacterium to transcribe high levels of a human gene. Both a and b present problems.

SYNTHESIZE

1. You set up four RT-qPCR reactions all containing the same amount of total RNA. When the reactions are complete, you see that you have 10,000 units of fluorescence after 20 cycles for sample A, after 25 cycles for sample B, after 18 cycles for sample C, and after 22 cycles for sample D. Which of the samples had the most copies of target mRNA at the start of the RT-qPCR? a. b.

3. What potential problems must be considered in creating a transgenic bacterium with the human insulin gene isolated from genomic DNA to produce insulin?

DNA primers are required. DNA polymerase is stable at high temperatures. Ligase is essential. dNTPs are necessary.

1. Many human proteins, such as hemoglobin, are only functional as an assembly of multiple subunits. Assembly of these functional units occurs within the endoplasmic reticulum and Golgi apparatus of a eukaryotic cell. Discuss what limitations, if any, exist to the large-scale production of genetically engineered hemoglobin. 2. Amyloid beta is a proteolytic product of a protein found in the brain called amyloid precursor protein (APP). At high concentrations, amyloid beta clumps together and may be a cause of Alzheimer disease. How might you go about studying, in a mouse model, whether particular mutations maintained the normal function of APP, while preventing clumping of the amyloid beta derived from APP?

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18 CHAPTER

Genomics Chapter Contents 18.1

Mapping Genomes

18.2

Sequencing Genomes

18.3

Genome Projects

18.4

Genome Annotation and Databases

18.5

Comparative and Functional Genomics

18.6

Applications of Genomics

©William C. Ray, Director, Bioinformatics and Computational Biology Division, Biophysics Program, The Ohio State University

Introduction

Visual Outline Genomics includes Mapping genomes types

Genetic maps

Physical maps

relative distance

absolute distance

Sequencing genomes allows by

Genome analysis

Genome annotation

Genome projects

includes

includes Open reading frames

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Class of DNA includes

Gene markers

Comparative genomics

Dideoxy sequencing Next-gen sequencing

Ric e garcane C orn

W h e at

Functional genomics

Proteomics Proteincoding DNA

Non-coding DNA

The pace of discovery in biology in the last 30 years has been dramatic. Starting with the isolation of the first genes in the mid-1970s, researchers obtained the first complete genome sequence, that of the bacterium Haemophilus influenza (shown in the diagram above with genes of similar function similarly colored), by the mid-1990s. A complete draft sequence of the human genome had been completed by the turn of the 21st century. Put another way, science moved from cloning a single gene, to determining the sequence of a million base-pairs in 20 years, to determining the sequence of 3 billion base-pairs in five more years, to now being able to sequence 20 billion basepairs in a few hours for as little as $1000. In chapter 17, you learned about biotechnology, and in this chapter you will see how some of those technologies have been applied to the analysis of whole genomes. Genomics integrates classical and molecular genetics with biotechnology to study the structure, function, and relatedness of genomes.

18.1 Mapping

Genomes

Learning Outcomes 1. Distinguish between a genetic map and a physical map. 2. Describe the pros and cons of restriction mapping, chromosome mapping, and mapping by STS. 3. Relate the information from a genetic map to the information from a physical map.

We use maps to find locations, and depending on the nature of the location we use different types of maps: a map of the world, a map of a country, or street maps of a city. In genomics, a gene can be located to a chromosome, to a subregion of a chromosome, and finally to a precise location in the sequence of chromosomal DNA. A  map at the DNA sequence level requires knowing the entire sequence of the genome, once technologically impossible. Knowing the entire sequence of a genome is useless, however, without knowledge of how a genetic region affects particular phenotypes. This sort of information resides in genetic maps. To make an analogy from geographic maps: finding a single gene within the sequence of the human genome is like trying to find a house on a map of the world.

family relationships and pedigrees, and use statistical analysis to determine recombination frequencies. Distances on a genetic map are measured in centimorgans (cM) where 1 cM corresponds to 1% recombination frequency between two loci. Two markers 1 cM apart are relatively close together as there is only a 1% chance that they will be separated during meiosis. Early human genetic maps consisted of linked genes for disease susceptibility that had been mapped to specific chromosomes, but no overall map. This did not change until the advent of molecular markers that did not cause detectable phenotypes (see chapter 13). Not only does this provide many polymorphic markers, but they can be readily integrated with molecular maps. The human genetic map is now quite dense, with a marker roughly every 1 cM. Genetic maps are very important because they locate genetic traits on a chromosome, but they also have limitations. First, the distribution of crossover, or recombination, events is not random. In fact, there are now extensive data indicating the presence of hot spots for recombination in humans, mice, and most other organisms examined. Additionally, recombination frequencies can vary between individuals, and are affected by chromatin structure. All of this means that the relationship between genetic distance (in centimorgans), and actual physical distance (in base-pairs [bp]) varies across the genome. But, with a complete genome sequence and a dense genetic map, they can still be superimposed to provide complementary views of the genome.

Genomics uses many approaches to analyze entire genomes

Physical maps provide absolute distances between genetic markers

We construct different kinds of maps of genomes from different kinds of data, and for different kinds of analysis. Fundamentally, there are two kinds of maps: genetic maps and physical maps. Both of these types of maps use genetic markers, which can be any detectable differences between individuals. Genetic maps are abstract maps that place the relative location of genetic markers on chromosomes based on recombination frequency (see chapter 13). Physical maps precisely position genetic markers in the genome, with the ultimate physical map being the complete DNA sequence of a genome. The field of genomics unifies many different types of maps, or views, of the genome to facilitate large-scale analysis. Although not technically the study of the genome, the relationship between the genome and the expressed RNAs, and between the genome and the expressed proteins, can be studied. Genomic studies have shed light on evolutionary relationships, genetic susceptibility to disease, and development.

Unlike a genetic map, which provides the relative position of a marker in the genome, a physical map provides the absolute location of a marker. The first physical maps used the early tools of molecular biology: restriction enzymes. As technologies became more sophisticated, so too did the nature of physical maps. The ultimate form of a physical map is the placement of many genetic markers on the complete DNA sequence of a genome. Distances between markers on a physical map are measured in base-pairs (1000 base-pairs [bp] equal 1 kilobase [kb]). Segments of DNA can be physically mapped without knowing the DNA sequence, or whether the DNA encodes any genes. In fact, many genome sequencing projects begin by constructing physical maps. There are three general types of physical maps: (1) restriction maps, constructed using restriction endonucleases; (2) chromosome maps; and (3) sequence-tagged site (STS) maps.

Genetic maps provide relative distances between genetic markers

Restriction maps are generally not suitable for DNA molecules greater than about 50 kb, which means that they are only useful for some organelle and some viral genomes. The first physical maps were created by cutting DNA with single restriction enzymes and with combinations of restriction enzymes (figure 18.1). The analysis of the patterns of fragments generated is used to generate a map. Restriction mapping can be applied to larger genomes if the DNA is first fragmented into smaller pieces. In terms of larger pieces of DNA, this process is repeated and then used to put the pieces back together, based on size and overlap, into a contiguous segment of the genome, called a contig.

Genetic maps, also called linkage maps, use the process of recombination during meiosis, which alters the linkage relationships of alleles on chromosomes (see chapter 13). These “genetic markers” can be genes, as detected by phenotypic differences, or differences in DNA sequences that can be detected by PCR or restriction endonuclease digestion, as described in chapter 17. Classical genetic mapping uses controlled crosses to determine the number of recombinant progeny for specific pairs of markers. Obviously, this is not practical in humans, so we use pre-existing data in the form of

Restriction maps

chapter

18 Genomics 369

+B

enzyme B

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1. Multiple copies of a segment of DNA are cut with restriction enzymes.

Ph1 22

Molecular weight marker 2. The fragments produced by enzyme A only, by enzyme B only, and by enzymes A and B together are run side-by-side on a gel, which separates them according to size.

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4. A physical map is constructed.

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a. Karyotype of human chromosomes showing the translocation between chromosomes 9 and 22. b. FISH using a bcr (green) and abl (red) probe. The yellow color indicates the fused genes (red plus green fluorescence combined). The abl gene and the fused bcr-abl gene both encode a tyrosine kinase, but the fused gene is always expressed.  ©W. Achkar, A. Wafa, H. Mkrtchyan, F. Moassass and T. Liehr, “Novel

9 kb

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complex translocation involving 5 different chromosomes in a chronic myeloid leukemia with Philadelphia chromosome: a case report,” Molecular Cytogenetics, 2: 21 (2009). doi:10.1186/1755-8166-2-21

A 10 kb

19 kb

Figure 18.1 Restriction enzymes can be used to create a physical map. DNA is digested with two different restriction enzymes singly and in combination. The DNA fragments are separated based on size using gel electrophoresis. The location of sites can be deduced by comparing the sizes of fragments from the individual reactions with those from the combined reaction.

Chromosome maps Cytologists studying chromosomes with light microscopes found that by using different stains, they could produce reproducible patterns of bands on the chromosomes. In this way, they could identify all of the chromosomes and divide them into subregions based on banding pattern. The use of different stains allows for the construction of a cytological map of the entire genome. These maps break each chromosome into a “p arm” and a “q arm” and then break these into regions and subregions based on banding patterns. All later maps have been correlated with this lowresolution map. These large-scale physical maps are like a map of 370 part III Genetic and Molecular Biology

Interphase nucleus of leukemic cell containing the Philadelphia chromosome (Ph1)

Figure 18.2 Use of fluorescence in situ hybridization (FISH) to correlate cloned DNA with cytological maps. 

14 kb

B

A

fused genes

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A

2 kb 3 kb

abl (on normal 9)

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3. The fragments are arranged so that the smaller ones produced by the simultaneous cut can be grouped to generate the larger ones produced by the individual enzymes.

Reciprocal translocation between one 9 and one 22 chromosome forms an extra-long chromosome 9 (“der 9”) and the Philadelphia chromosome (Ph1) containing the fused bcr-abl gene. This is a schematic view representing metaphase chromosomes.

?

Inquiry question How can you explain the pattern of red, green, and yellow dots seen in the right panel of figure 18.2b?

an entire country in that they encompass the whole genome but do so at low resolution. Cytological maps are used to characterize chromosomal abnormalities associated with human diseases, such as chronic myelogenous leukemia. In this disease, a reciprocal translocation occurs between chromosome 9 and chromosome 22 (figure 18.2a), resulting in an altered form of tyrosine kinase that is always turned on, causing white blood cell proliferation and, consequently, leukemia. Fluorescent in situ hybridization, which we discussed in chapter 17, can also be used to generate chromosomal maps (figure 18.2b). Initially the level of resolution of FISH was approximately 1 Mb (megabase); this means that two points on a chromosome closer than 1 Mb could not be distinguished as separate points. This limitation made FISH really useful only for determining whether a particular DNA sequence was found on a particular chromosome. Technical advances in the 1990s allowed the use of interphase chromosomes as well as metaphase chromosomes, and

STS sites

STS 1

STS 2

STS 3

STS 1

STS 4

STS 2

Clone A

DNA PCR primers 1. The location of 4 STSs in the genome is shown. PCR is used to amplify each STS from different clones in a library. Amplifying each STS by PCR generates a unique fragment that can be identified.

Clone B

STS 4

Clone C

Clone D

STS 2

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STS 3

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Clone C Longer fragments

STS 3

Clone D STS 1

STS 2 STS 1

STS 3

Clone B

PCR runs with four clones Clone A

STS 2

Shorter fragments

2. The products of the PCR reactions are separated by gel electrophoresis producing a different-sized fragment for each STS.

STS 2

Contig 3. The presence or absence of each STS in the clones identifies regions of overlap. The final result is a contiguous sequence (contig) of overlapping clones.

Figure 18.3 Creating a physical map with sequence-tagged sites. The presence of landmarks called sequence-tagged sites, or STSs, in the human genome made it possible to begin creating a physical map large enough in scale to provide a foundation for sequencing the entire genome. (1) Primers (green arrows) that recognize unique STSs are added to cloned DNA, followed by DNA amplification via polymerase chain reaction (PCR). (2) PCR products are separated based on size on a DNA gel, and the STSs contained in each clone are identified. (3) Cloned DNA segments are aligned based on STSs to create a contig.

Data analysis You could construct the physical map using a subset of the clones in panel 2. List the possible combinations that would allow you to construct the physical map with the fewest number of clones.

this improved the resolution of FISH to 25 kb. This advance meant the technique could be used for finer mapping.

Sequence-tagged site maps Restriction mapping allows relatively high-throughput, technically easy, high-resolution mapping of relatively small DNA molecules. FISH, on the other hand, provides low-resolution mapping of large DNA molecules and is technically challenging. Sequence-tagged site (STS) mapping provides a powerful alternative that combines the best of restriction mapping with the best of mapping via FISH. STS mapping allows the rapid construction of high-resolution physical maps of large DNA molecules with few technical challenges. An STS is a short, unique stretch of genomic DNA that can be amplified by PCR (see chapter 17). STS mapping asks whether two STSs are on the same DNA molecule. This involves randomly fragmenting DNA into overlapping fragments. If two STS markers are commonly found together on fragments of DNA, then they are relatively close together. If they are not commonly found together on fragments of DNA, then they are likely to be farther apart (figure 18.3). Much as genetic mapping is based on the frequency with which

markers are separated by recombination, STS mapping is based on the frequency with which markers can be separated by breaking DNA into fragments. Markers closer together will be separated less frequently than markers that are farther apart. The farther apart two markers are, the more likely a break can occur between them. Fragments of DNA can be pieced together using the STSs by identifying overlapping regions in fragments. Because of the high density of STSs in the human genome and the relative ease of identifying an STS in a DNA clone, investigators were able to develop physical maps on the huge scale of the 3.2 billion bp genome in the mid-1990s (figure 18.3). STSs provide a scaffold for assembling genome sequences.

Physical maps can be correlated with genetic maps Genetic maps can identify relevant regions of the genome that affect specific phenotypes, but they tell us nothing about the nature of these “genes.” The correlation of physical maps with genetic maps allows us to find the sequence of genes that have been mapped genetically. chapter

18 Genomics 371

Genes

1

2

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5

Genetic map created by analyzing patterns of inheritance Markers

1

2 3

4

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Physical map created by analyzing cloned DNA

The problem in finding genes is that the resolution of genetic maps is at present not nearly as good as the resolution of the genome sequence. In the human genome, markers that are 1 cM apart may be as much as a million base-pairs apart. Because the markers used to construct genetic maps are now primarily molecular markers, they can be easily located within a genome sequence. Similarly, any gene that has been cloned can be placed within the genome sequence and can be mapped genetically. This provides an automatic correlation between the two maps. Genes that have been mapped genetically can, in theory, be readily located within the DNA sequence of a genome. In practice, this can be challenging as the relationship between genetic distance and physical distance varies across the genome. So 1 cM of genetic distance can actually be composed of different numbers of base-pairs in different regions of a genome.

The ultimate physical map is the base-pair sequence of an entire genome. Large-scale, high-throughput DNA sequencing has made whole-genome sequencing realistic for smaller labs and biotechnology companies. Conceptually, all approaches to sequencing DNA adapt DNA replication to an in vitro environment. Older approaches use modified nucleotides to stop or pause replication and limit the replication to just one strand of the DNA. The rapid advances in genomics have been enabled by newer and much faster automated sequencing technologies.

Dideoxy terminator sequencing remains important in genome sequencing In the last 10 years there have been huge improvements to the technologies used to sequence DNA. There are still situations, however, where the original DNA sequencing methods are useful. One original method for sequencing DNA developed by Fred Sanger in the 1970s mimics DNA replication. This technique depends on the use of dideoxynucleotides in DNA sequencing reactions. The dideoxynucleotides act as chain terminators, and halt replication when they are incorporated. All DNA nucleotides lack a hydroxyl group at the 2′ carbon of the sugar, but dideoxynucleotides also have no 3′ OH group. Recall from chapter 14 that each additional base is added to the growing 3′ end of a strand of DNA by a reaction with the OH group of the previous nucleotide to the triphosphate of the next nucleotide (see figure 14.12). A dideoxynucleotide can be incorporated, but then cannot be added to, as it lacks the 3′ OH, and thus terminates the growing chain.

Learning Outcomes Review 18.1

Maps of genomes can be either physical maps or genetic maps. Physical maps include cytogenetic maps of chromosome banding, or restriction maps. Genetic maps are correlated with physical maps by using DNA markers such as sequence-tagged sites (STSs) unique to each genome. While genetic maps position markers, such as genes, relative to one another, physical maps position them at a specific genetic location on a sequence of DNA. Genetic and physical maps are used to aid in the sequencing phase of genome projects. ■■

How could it be that two markers on a genetic map are 10 cM apart, while two markers in a different part of the same genetic map are 12 cM apart, but both sets of markers are separated by the same amount of DNA?

18.2 Sequencing

Genomes

Learning Outcomes 1. Discriminate between dideoxy terminator sequencing and next-generation sequencing technologies. 2. Compare and contrast clone-contig and shotgun methods of genome sequencing and assembly.

372 part III Genetic and Molecular Biology

N

NH2 N

O −O

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Dideoxy terminator sequencing was originally performed manually where the experimenter would perform four separate reactions, each with a single dideoxynucleotide and the four deoxynucleotides. When a dideoxynucleotide is incorporated, it will terminate synthesis at a specific base. Thus, each reaction produces a set of nested fragments that each end in a specific base, and the four reactions together represent fragments that terminate at each possible base. The fragments were separated by high-resolution gel electrophoresis, which can separate fragments differing in length by a single base. This produces a ladder of fragments that allow the sequence to be read from bottom (smallest fragment) to top (longest fragments). To automate this, each dideoxynucleotide is labeled with a fluorescent dye, and all four are added to a single DNA synthesis reaction. As before, the random incorporation of dideoxynucleotides produces a set of fragments that end in specific bases, but in this case, each base is represented by a color. The dye-labeled

DNA in a single day. In some ways, this was the first step in creating the massively parallel sequencing platforms that characterized the next generation of sequencing technologies.

Automated Enzymatic DNA Sequencing Template DNA polymerase 3′

Next-generation sequencing uses massively parallel technologies to increase throughput

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Figure 18.4 Automated dideoxy DNA sequencing. 

The sequence to be determined is shown at the top as a template strand for DNA polymerase with a primer attached. In automated sequencing, each ddNTP is labeled with a different-colored fluorescent dye, which allows the reaction to be done in a single tube. The fragments generated by the reactions are shown. When these are electrophoresed in a capillary tube, a laser at the bottom of the tube excites the dyes, and each will emit a different color that is detected by a photodetector.

nested fragments are separated in a capillary tube by electrophoresis and as each molecule passes a laser, it fluoresces. The nucleotide terminating the DNA strand is interpreted based on the color of the fluorescence and automatically reported to a computer (figure 18.4). Although this approach is still labor intensive, automation allows thousands of sequencing reactions to be performed in parallel. This means it is possible to sequence nearly 900 kb of

Next-generation sequencing (NGS) technologies first appeared 10 or so years ago. Since then, there have been huge improvements in the speed of sequencing, in the cost per sequenced base, and in the length of sequence read per sequencing reaction. The result is a staggering increase in the number of genomes sequenced and resequenced in the past 10 years. In the future, this abundance of data could be translated into discoveries that reveal novel evolutionary relationships, improve the personalization of health care, and improve diagnostic medicine. There are numerous NGS technologies in the market, but all have some common features. First, in contrast to dideoxy sequencing, DNA can be sequenced without a need to first construct a genomic library by conventional cloning (see chapter 17). Second, instead of thousands of reactions being prepared and analyzed simultaneously, up to millions of sequencing reactions can be performed simultaneously; it is this feature of the technology that has led to the term “massively parallel.” Third, the time-consuming electrophoresis previously required is not needed. Instead, sequencing reactions occur simultaneously in solution and are read directly by the sequencing equipment (figure 18.5). Because of the increase in sequencing speed and the reduction in cost, some bacterial genomes can be sequenced in as little as a few hours! Although next-generation sequencing has produced dramatic reductions in cost, and produces much larger amounts of data, there is a down side. While these technologies allow sequencing of many molecules simultaneously, they produce less information per molecule sequenced. That is, the read length tends to be short, on the order of 50 bp up to 1000 bp for most next-generation sequencing methods. This places even more emphasis on computing power for accurate genome assembly. This has led to newer assembly algorithms, but even these struggle when there is a lot of repetitive DNA combined with short reads. There are some even newer technologies that produce much longer reads, even up to tens of thousands of bp. The down side of these technologies is that they tend to be more error prone. An evolving use of these methods is to combine longer, less accurate reads with many shorter, more accurate reads. In 2014, it was reported that a human genome had been sequenced for $1000. This represents a cost reduction of nearly 10,000-fold relative to the initial cost of sequencing a human-sized genome approximately 10 years before that.

Sequenced genome fragments are assembled into complete sequences The most complex task in a large-scale sequencing project is the assembly of individual pieces of sequence information into a complete genome. A genome is first fragmented into overlapping pieces that are sequenced, but then must be reassembled by matching short overlapping regions. The shorter the sequenced fragments, and the more repetitive DNA present in the genome, the harder it is to piece together the puzzle. Two strategies can be employed: (1) assemble portions of chapter

18 Genomics 373

Single DNA molecules are attached to a solid surface

DNA molecules are copied using PCR, amplifying the template

Shotgun assembly

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A benefit of shotgun assembly is that it does not rely on any genetic or physical maps. This means that no prior information about the sequenced genome is required. The first genome ever sequenced, the genome of the bacterium Haemophilus influenzae, was sequenced and then assembled using the shotgun approach. The genome of the bacterium was broken into fragments approximately 1.5 to 2 kb in length that were cloned into suitable vectors (see chapter 17). The ends of the fragments were sequenced and then, based on overlaps in the sequence, the pieces of DNA sequence could be stitched back together to form the whole 1.8-Mb genome (figure 18.6b). This strategy rarely gives a completely assembled genome sequence. A

G

Remove unincorporated nucleotides

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bled genomic sequence. A genome is fragmented into larger fragments—approximately 1 to 1.5 Mb. Then, those fragments are mapped into a larger contiguous segment of DNA, called a contig, using genetic markers such as STSs or restriction endonuclease sites. The fragments of DNA 1 to 1.5 Mb (megabases) in size can be sequenced and assembled using the shotgun approach.

G

Reverse termination (chemically or enzymatically) Repeat cycle adding additional nucleotides

Figure 18.5 Illumina next-generation DNA sequencing. In Illumina next-generation sequencing, the template

DNA molecules are attached to a solid surface and amplified by PCR to create islands of DNA. Four fluorescently labeled terminator nucleotides are then added, and after one is incorporated into new DNA, synthesis stops. The color of the incorporated nucleotide is read, and then the nucleotide is chemically altered so that another nucleotide can be added. This process of single-nucleotide addition is repeated to read the sequence of the DNA in each island.

a chromosome first, and then figure out how the bigger pieces fit together (clone-contig assembly); or (2) try to assemble all the pieces at once, instead of in a stepwise fashion (shotgun assembly).

Clone-contig assembly Clone-contig assembly methods (figure 18.6a) are used to assemble the pieces of sequenced DNA from more complex genomes such as those of eukaryotes. Unlike shotgun assembly, this kind of method requires a physical map to build the completely reassem374 part III Genetic and Molecular Biology

1. Large DNA clones are first isolated. These are arranged into contiguous sequences based on overlapping tagged sites. 2. Large clones are fragmented into smaller clones for sequencing. 3. The entire sequence is assembled from the overlapping larger clones.

a. Shotgun Method 1. Cut DNA of entire chromosome into small fragments and clone.

2. Sequence each segment and arrange based on overlapping nucleotide sequences.

b.

Figure 18.6 Comparison of sequencing and genome assembly methods. a. The clone-contig method uses large clones

assembled into overlapping regions by STSs. Once assembled, these can be fragmented into smaller clones for sequencing. b. In the shotgun method, the entire genome is fragmented into small clones and sequenced. Computer algorithms assemble the final DNA sequence based on overlapping nucleotide sequences.

variety of other approaches are used to close the gaps that become obvious only when the sequences have been assembled.

Learning Outcomes Review 18.2

Because of the enormous size of some genomes, sequencing requires the use of automated sequencers running many samples in parallel. Two approaches have been developed for whole-genome sequencing and assembly: one that uses clones already aligned by physical mapping (clone-contig sequencing and assembly), and one that involves sequencing random clones and using a computer to assemble the final sequence (shotgun sequencing and assembly). In either case, significant computing power is necessary to assemble a final sequence. ■■

What are the pros and cons of the different approaches to whole-genome sequencing and assembly?

18.3 Genome

Projects

Learning Outcomes 1. Describe the findings of the Human Genome Project. 2. Explain why sequencing the human genome was a relatively simple endeavor compared to sequencing the wheat genome. 3. Describe the amount and kind of variation in the human genome.

Automated-sequencing technology has produced huge amounts of sequence data. This has allowed researchers studying complex problems to move beyond approaches restricted to the analysis of individual genes. Sequencing the genome of any species is a form of descriptive science that does not shed light on genome organization, gene function, or how these may be interrelated, but it does provide the groundwork for these studies.

using just shotgun sequencing and better assembly software. Although there is some truth to this, Venter’s company, now called Celera Genomics, also had the publicly available physical maps from the HGP, so they were not using a pure shotgun approach. The approach worked for the bacterium Haemophilus influenzae but this genome is orders of magnitude smaller, and less complex than the human genome. In June 2000, a draft sequence of the entire genome was jointly announced by the HGP and Celera. This draft sequence contained many gaps and errors, but represented a functional, albeit incomplete, version of the human genome. In 2003, the draft sequence was replaced by the first reference sequence. The difference between these is that the reference sequence has fewer sequencing errors, fewer gaps, and greater coverage, and attempts to take variation into account. The initial reference sequence still contained 150,000 gaps, and consisted of 2.69 Gb of sequence. These data are curated by the Genome Reference Consortium, an international consortium, with reference sequences named GRCh plus a number. We are up to GRCh38 which consists of 3.1 Gb of sequence and only 350 gaps, and also includes 360 regions with alternative loci. One obvious question about the reference genome is, whose genome is it? In fact, the original source of DNA came from 20 volunteers. Later analysis showed that 70% of the sequence is from a single donor, 23% is from 10 individuals, and 7% from over 50 DNA libraries. Work is ongoing to sequence genomes from diverse populations to create a “pan genome” that better reflects actual human diversity. The first significant result to come out of the Human Genome Project was the discovery that the number of genes, long estimated to be around 100,000, was actually only about 20,000. This is about 1.5 times as many genes as the fruit fly, and nearly half as many genes as rice (figure 18.7). Humans, mice, and puffer fish all have about the same number of genes. Clearly, organismal complexity is not a simple function of either genome size or gene number.

Data analysis If the human genome contains

approximately 3 billion base-pairs, 20,000 genes, and only 1% of the genome codes for genes, what is the approximate length of a gene?

The Human Genome Project has sequenced and mapped most of the human genome

Sequencing many human genomes reveals enormous variation

The Human Genome Project (HGP) officially began in 1990, but it arose from the advances in molecular biology in the previous decade. While the actual human genome sequence is most familiar to the general public, the creation of genetic and physical maps were critical parts of the project. A physical map covering 94% of the genome, with markers about every 200 kb, was completed by 1995. A genetic map with markers about every 1.6 cM was completed in 1996. Since these early maps, the number of markers and the resolution have increased significantly. The project drove advances in automation that increased the speed and reduced the cost of sequencing. Ultimately, this formed the basis for the clonecontig and shotgun approaches used by the HGP. In 1998 a competitor from the private sector emerged when Craig Venter, who founded The Institute for Genomic Research, claimed that they could sequence the genome faster and cheaper

With this reference genome “in hand,” one question that might occur to you is: “How similar is my genome to the reference genome?” A this point, we have sequenced literally thousands of individual genomes, which has revealed the enormous diversity in human genomes. In chapter 15, we explored the different kinds of genetic variation; now we will examine the extent of this variation.

Single-nucleotide variation (SNV) There are now over 650 million SNV entries in a database devoted to genetic variation. A genome selected at random, perhaps yours or mine, would be expected to differ from the reference sequence at about 4 million sites. Small insertions or deletions of from 1 to 49  bp (indels) are another large source of variation. Our random genome would differ from the reference at 400,000 sites with indels. There is also massive variation in short repetitive regions that are chapter

18 Genomics 375

Estimated Number of Genes

40,000

prokaryotes eukaryotes

30,000

Thale cress (Arabidopsis thaliana)

Puffer fish (Fugu rubripes) Human Mouse (Mus (Homo sapiens) Fission yeast (Schizosaccharomyces pombe) musculus) Nematode Bakers yeast (S. cerevisiae) Mosquito (Anopheles sp.) (Caenorhabditis Fruit fly (Drosophila melanogaster) Protozoan elegans) (Encephalitozoon Malaria microbe (Plasmodium falciparum) cuniculi ) Slime mold (Dictyostelium discoideum)

20,000 10,000 0

Figure 18.7 Size and complexity of genomes. 

Rice (Oryza sativa)

0

1

10

100

1000

Size of Genome (million base-pairs) Log Scale

difficult to sequence accurately. We will consider these in the next section when we explore the nature of the genome in more detail.

Structural variation (SV) Insertions or deletions of more than 50 bp, and more complex rearrangements, are called structural variation (SV). Deletion is the most common form of SV, with our random genome and reference genome differing at around 5000 sites due to deletion. Duplication accounts for another 1000, and insertion for 1500 differences. These also involve the loss or gain of DNA and are thus considered copy number variation (CNV). A special case of insertions involves mobile genetic elements (MEI), discussed in section 18.4. Comparing our random genome to the reference reveals about 2000 different MEI. Balanced rearrangements that do not affect copy number also occur. These are primarily inversions of a region, with about 40 of these in our genome comparison. Even more rare, but causal in several cancers, are reciprocal translocations where material is swapped between different chromosomes.

Population-level variation So what does all of this variation tell us about ourselves? The majority of variants in a population are rare, but individuals in the population also carry many common variants. This implies that the rare variants that are prevalent in a population are due to new mutations that arise randomly. This pattern is consistent with humans sharing a demographic history that goes back 150 to 200 thousand years. Some subpopulations, including from Europe, Asia, and the Americas, appear to have had bottlenecks, with population sizes in the low thousands around 15 to 20 thousand years ago. Variants that were fixed in those small populations are therefore common in the population, and as population sizes increased, random mutation produced a large number of rare variants.

The Wheat Genome Project illustrates improvements in genome assembly Wheat, Triticum aestivum, is used to feed about 30% of the world’s population. Better understanding wheat genomics can inform breeding practices to increase yield and improve drought and disease resistance. It can also provide insight into the evolution of wheat species. Given expanding populations, dwindling water resources, and global climate change, applying modern genetics to the improvement of crop plants will be critical to maintain secure global food supplies. 376 part III Genetic and Molecular Biology

10,000

In general, eukaryotic genomes are larger and have more genes than prokaryotic genomes, although the size of the organism is not the determining factor. The mouse genome is larger than the human genome and the rice genome is larger than both human and mouse genomes.

Genetically, wheat is an allohexaploid (see chapter 24) produced by two hybridization events. This means the wheat genome contains the contributions of three diploid ancestors designated A, B, and D, each containing about 5 billion bp of DNA. The modern wheat genome has 21 chromosomes, 7 from each ancestral genome, and an estimated size of 16 Gb. There are two main challenges to sequencing and assembling the wheat genome: (1) its large size, and (2) a large amount of repetitive DNA, including nearly identical sequences scattered throughout due to the polyploid nature. A first attempt to sequence the wheat genome in 2012 assembled only 5.4 Gbp, or about one-third of the genome. A second attempt published 2 years later attempted to sequence a chromosome at a time, yielding a genome assembly of 10.2 Gbp, or about two-thirds of the genome. In early 2017 a third attempt was produced that covered 78% of the genome. An additional problem with all 3 of these assemblies was that the length of contigs (see section 18.2) was relatively short. Finally, later in 2017, the most complete sequence yet was published; it covers 15.3 Gbp, with contigs more than 10 times longer than the previous best effort. This was accomplished by combining newer sequencing technology that produces very long reads, but with a high frequency of errors, with multiple runs with more accurate short reads. A similar approach was used to produce the first sequence of Aegilops tauschii, the species that contributed the “D genome” in the last hybridization event leading to modern wheat. The Ae. tauschii genome has a high degree of dispersed repeated sequences, including transposable elements that make up 84% of the genome. The Ae. tauschii genome appears to be accumulating large-scale rearrangements faster than other cereal genomes, probably by recombination between these repetitive elements.

Learning Outcomes Review 18.3

The Human Genome Project produced molecular and genetic maps, and a draft sequence followed by the reference sequence of the human genome. As we have sequenced many individuals, this has revealed enormous variation. The average genome differs from the reference in more than 4 million sites. These data also indicate that humans began from small populations that have recently expanded rapidly. The wheat genome project illustrates better assembly methods. ■■

Why are longer sequencing reads helpful with complex genomes like wheat?

18.4 Genome

Annotation and Databases

Learning Outcomes 1. Explain why we need to annotate genomes. 2. Compare and contrast the types of DNA found in genomes. 3. Evaluate the conclusions of the ENCODE project.

While laborious to produce, a genome sequence itself is of little intrinsic interest. More important is the number and kind of genes present in the genome, and how these genes contribute to phenotype. We use the process of genome annotation to attach this kind of information to the actual genome sequence in databases.

Genome annotation assigns function to DNA sequences in genomes An annotation is a record attached to a DNA sequence held in a database that identifies the DNA sequence as a gene. The record can contain information about the gene, its structure and function, and the product it ultimately produces (RNA or protein). This may also include evidence supporting the identified gene function. This is intended to provide biological relevance to otherwise meaningless sequence data. Some forms of genome annotation involved automated computer searches that are checked as needed by experts. This is possible because we know the “signature” for sequences that are associated with genes such as promoters and other regulatory regions. The computer can search for a start codon, followed by codons that encode amino acids, and eventually a stop codon. Such a sequence is called an open reading frame (ORF). Intron/exon structure can be predicted based on splicing signals, allowing the prediction of possible mRNAs and the amino acid sequences of encoded proteins.

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search for similar sequences already residing in GenBank. Significant sequence similarity with other genes or gene products in a different species could suggest conservation of function. These are just a small part of the expanding field of bioinformatics.

Expressed sequence tags identify genes that are transcribed Another way to identify sequences that encode proteins is to isolate total mRNA from different tissues, convert this to cDNA for sequencing, and then map the cDNA onto the genomic sequence (figure 18.8). This process can be simplified by just sequencing one or both ends of as many cDNAs as possible. These short sections of cDNA are sufficient for identification, and have been named expressed sequence tags (ESTs). An EST is another form of STS that can be included in physical maps.

The majority of the human genome does not encode protein DNA sequences that are used to produce a protein are called coding sequences (CDS), and they are annotated in databases as CDS. The genome also encodes a number of functional RNAs that are never translated. These include tRNA, rRNA, snRNAs, and miRNA, which are all involved in the process or control of gene expression. This still represents a small fraction of the total DNA in a genome. So how do we characterize the rest of the genome? As the amount of genomic information has increased, the number of different ways to characterize the genome has also increased. Below is one simple way of broadly characterizing different “kinds” of DNA in the genome.

Noncoding DNA in eukaryotes A notable characteristic of eukaryotic genomes is the fraction of DNA that does not encode protein. The Human Genome Project revealed a particularly startling picture: each of your cells contains nearly 2 m of DNA, but only about 2.5 cm of that DNA encodes proteins! Nearly 99% of the DNA in your cells is noncoding DNA.

Inquiry question Given the sequence of a genome,

how might you predict the number of proteins produced by a genome? Why might your estimate be inaccurate?

Inferring function across species: the BLAST algorithm Once a potential gene or gene product has been identified, one way to infer function is to search for similar sequences in other species. The most common tool for this is a search algorithm called the Basic Local Alignment Search Tool (BLAST). This kind of search is possible because of large public databases containing all known gene and genome sequences. The database GenBank, maintained by the National Center for Biotechnology Information (NCBI) in the U.S., is a repository for all public DNA sequence data. The August 2020 release contains a staggering 9.8 × 1012 bp. Similar large primary public databases are maintained by the National Institute of Genetics in Japan, and the European Bioinformatics Institute. Using a networked computer, any user can submit potential gene or gene product sequences to the BLAST server, which will

Exon Genomic structure

Exon

Exon Intron

5’ 5’ UTR

3’ UTR

ORF cDNA

5’ 5’ UTR

3’ UTR

3’

Poly-A tail Examples of ESTs

5’ 5’ 3’ 3’

Figure 18.8 ESTs can be used to map expressed genes onto the genome. Sequencing the end of ESTs produces sequence data that can be aligned with predicted cDNA structure and mapped onto corresponding genomic structures. Through sequencing many ESTs, complete genomic structures can be determined. chapter

18 Genomics 377

TA B L E 1 8 .1

Classes of DNA Sequences Found in the Human Genome

Class

Description

Protein-encoding genes

Translated portions of the approximately 20,000 genes scattered about the chromosomes

Introns

Noncoding DNA that constitutes the great majority of each human gene

Segmental duplications

Regions of the genome that have been duplicated

Pseudogenes (inactive genes)

Sequence that has characteristics of a gene but is not a functional gene

Structural DNA

Constitutive heterochromatin, localized near centromeres and telomeres

Simple sequence repeats

Stuttering repeats of a few nucleotides such as CGG, repeated thousands of times

Transposable elements

21%: Long interspersed elements (LINEs), which are active transposons 13%: Short interspersed elements (SINEs), which are active transposons 8%: Retrotransposons, which contain long terminal repeats (LTRs) at each end 3%: DNA transposon fossils

Noncoding RNA

RNAs that do not encode a protein but have regulatory functions, many of which are currently unknown

Genes coding for proteins are scattered in clumps amongst the noncoding DNA in the human genome. Seven major types of noncoding DNA have been described in the human genome (table 18.1 shows the composition of the human genome, including noncoding DNA): Noncoding DNA within genes. As discussed in chapter 15, a human gene is made up of numerous pieces of coding DNA (exons) interspersed with lengths of noncoding DNA (introns). Introns comprise about 24% of the human genome, whereas the exons comprise less than 1.5%. Structural DNA. Some regions of the chromosome remain highly condensed, tightly coiled, and untranscribed. These regions, called constitutive heterochromatin, tend to be localized around the centromere or near the ends of chromosomes, at telomeres, and to contain highly repetitive DNA. Simple sequence repeats. There are two forms of tandem repeat variation that differ in size and are scattered within the genome. Short tandem repeats (STR) consist of repeat units of 2–6 bp. Variable number tandem repeats (VNTR) contain repeat units of of 7–49 bp. Together they make up about 3% of the genome. Segmental duplications. These are blocks of genomic sequences of 10,000 to 300,000 bp that have duplicated and moved either within a chromosome, or to a nonhomologous chromosome. Pseudogenes. These inactive genes may have lost function because of mutation. There are almost 15,000 characterized pseudogenes in the human genome. Transposable elements. Forty-five percent of the human genome consists of DNA sequences that can move from one place in the genome to another. Some of these sequences code for proteins that allow their movement, but many do not. Because of the significance of these elements, they are described in this section. MicroRNA genes (miRNA). Hidden in the noncoding DNA is a mechanism for controlling gene expression. Specifically, DNA that was once considered “junk” has been shown to encode miRNAs, which are processed after transcription to 378 part III Genetic and Molecular Biology

lengths of 21 to 23 nt. About 10,000 unique miRNAs have been identified that are complementary to one or more mature mRNAs. These miRNAs regulate some of the complex developmental processes in eukaryotes by forming complexes with protein that affect gene expression (see chapter 16). Long, noncoding RNA (lncRNA). In addition to the many smaller RNAs such as miRNAs that are not translated into protein but serve a regulatory role, tens of thousands of longer noncoding RNAs likely regulate gene expression. This recently discovered, hidden world of regulatory networks reveals a new level of complexity in the precise control of gene expression. Long, noncoding RNAs have important roles in physiology and development. Only approximately 200 long, noncoding RNAs have been functionally characterized and the biological functions, if any, of the others remain unclear.

Transposable elements: Mobile DNA Transposable elements, also called transposons and mobile genetic elements, are sequences of DNA able to move from one location in the genome to another. Transposable elements move around in different ways. In some cases, the transposon is duplicated, and the duplicated DNA moves to a new place in the genome. When this happens, the number of copies of the transposon increases. Other types of transposons are excised without duplication and insert themselves elsewhere in the genome. The role of transposons in genome evolution is discussed in chapter 24. Human chromosomes contain four types of transposable elements. About 21% of the genome consists of long interspersed elements (LINEs). These ancient and very successful elements are about 6000 bp long, and they encode the enzymes needed for transposition. LINEs encode a reverse transcriptase enzyme that can make a cDNA copy of the transcribed LINE RNA. The result is a double-stranded DNA fragment that can reinsert into the genome rather than undergo translation into a protein. Since these elements use an RNA intermediate, they are called retrotransposons.

Short interspersed elements (SINEs) are similar to LINEs, but they do not encode the transposition enzymes and so cannot move without using the transposition machinery of LINEs. Nested within the genome’s LINEs are over half a million copies of a SINE element called Alu (named for a restriction enzyme that cuts within the sequence). The Alu SINE is 300 bp and represents 10% of the human genome. An Alu SINE can use the enzymes of the LINE of which it is a part to jump to a new chromosome location. Alu sequences can also jump into coding DNA sequences, causing mutations. Two other sorts of transposable elements are also found in the human genome. First, about 8% of the human genome is made from a type of retrotransposon called a long terminal repeat (LTR). Although the transposition mechanism is a bit different from that of LINEs, LTRs also use reverse transcriptase to create double-stranded copies of itself that can integrate into the cell’s genome. Second, dead transposons occupy approximately 3% of the genome; these are transposons inactivated by mutation that can no longer move. 3% 8% 13% 55%

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21%

LINEs SINEs LTRs dead transposons remaining noncoding and coding DNA in human genome

Inquiry question How could you show whether a sequence of DNA was functional or not?

A biochemical signature appears when a protein binds to a DNA sequence, when chromatin bends to form long-range interactions, or when a DNA sequence exhibits a particular pattern of histone modification. Given that there is likely a significant difference between these functionalities in different cell types, ENCODE derived their 80% value by testing the genomes of 144 different cultured human cell lines for functional elements. Some criticism has been directed at ENCODE for their use of cultured cell lines and stem cell lines. These cell lines may not be reflective of the situation in the genome of cells of an organism and may be more transcriptionally active than cells in tissues. Critics also claim that there are likely to be significant differences between functional elements in different developmental stages of an organism and under different environmental conditions.

Biological function The functional definition used by ENCODE is not necessarily synonymous with biological function. A sequence of DNA that is

Nucleosome

Long-range chromatin interactions

The ENCODE project seeks to identify all functional elements in the human genome The presence of noncoding DNA in genomes has been known for many years, but the biological importance of this DNA remained unclear. Since no function was assigned, this noncoding DNA was often referred to as “junk DNA.” Extensive analyses of the noncoding DNA have led some to suggest that the term “junk DNA” should be reevaluated. It may be best to reframe the DNA of no known function as “nonfunctional DNA.” The Encyclopedia of DNA Elements (ENCODE) project is a collaborative effort to identify all functional elements in the human genome. The primary conclusion of this work was that 80% of the DNA sequences in the human genome are functional. Of that 80%, approximately 62% is defined as transcriptionally active, although the majority of the transcribed, or potentially transcribed, RNAs have no known function. There has been much debate in the scientific community regarding what these numbers actually mean in terms of biological function, and the ENCODE consortium has revised its estimate of functionality to somewhere between 20 and 80%.

ENCODE’s definition of function ENCODE’s definition of a functional element includes any DNA sequence that results in protein production, that is transcribed, or that has a distinct and reproducible biochemical signature (figure 18.9).

Histone

DNase I DNA hypersensitive methylation sites

Chromatin modification

Transcription-factor binding sites

DNA Transcription factor Transcription machinery

Long-range regulatory elements

Chromosome

Protein-coding and noncoding transcripts Promoter structure

Transcribed region

Figure 18.9 Functional DNA elements defined by ENCODE. ENCODE uses a biochemical signature definition of

function. Biochemical signatures include methylation patterns of DNA sequences, modifications of histones in chromatin, sensitivity to DNase I, which suggests regions of transcriptional activity, sites of transcription factor binding, sequences identified as promoters, production of coding or noncoding transcripts, long-range regulatory elements such as enhancers, and long-range chromatin interactions. chapter

18 Genomics 379

transcribed, and therefore biochemically functional according to ENCODE, may not produce an RNA with a function necessary for the organism. Also, just because one or several transcribed RNAs have a demonstrated biological effect, this does not mean that all transcribed RNAs have biological effects. A natural extension of the definition of functional element could include any DNA sequence that is replicated; given such a biochemical definition, 100% of the genome would be functional, as 100% of the genome is replicated during cell division.

Selected-effect function ENCODE’s conclusions have also drawn fire from some evolutionary biologists. Most evolutionary biologists, and many biologists in general, subscribe to a selected-effect definition of function. The selected-effect definition of function requires that the function of a characteristic is the one selected for by purifying selection. For example, the selected function of the mammalian lung is gas exchange. Selective pressures have shaped that function. The rising and falling of the chest during breathing is caused by the inflation and deflation of the lungs, but that rising and falling action has not been selected for as the lung’s function. If a genetic sequence has a particular function, this will eventually be destroyed by the accumulation of random mutations over time, unless this is counteracted by natural selection. The only way that a functional sequence is protected from being destroyed is if purifying selection acts to resist the accumulation of mutations that degrade the function. Many biologists would argue that only DNA sequences under purifying selection should be considered functional. Such sequences are more likely to be conserved in a specific lineage and are also more likely to be conserved in related species. Based on these criteria, comparative genomics studies suggest that as little as 5 to 15% of the genome is functional. Clearly, there are some conflicting perspectives on the topic of the functionality of DNA sequences in the human genome, and ENCODE’s calculation of 80% functionality remains debatable. This being said, the technologies and means of data collection advanced by the ENCODE effort significantly advance our potential for understanding functions of DNA sequences in genomes. Similarly, ENCODE’s data regarding the biochemical functions of noncoding DNA provide an impressive map that can be used by others to explore the biological relevance of the identified functional elements in the human genome.

Learning Outcomes Review 18.4

Once a genome has been sequenced, assembled, and deposited into a database, functional elements are annotated. Functional elements can be identified by inference using the BLAST program to search related genes of known function. Only a fraction of the DNA in a genome contains information to make proteins. There are many variations on noncoding DNA, some with known function, much with no known function. The ENCODE project argues that most noncoding DNA is functional. This argument depends on how you define function. ■■

What explanation could you suggest, based on principles of natural selection, for the many repeated transposable elements in the human genome?

380 part III Genetic and Molecular Biology

18.5 Comparative

Genomics

and Functional

Learning Outcomes 1. Explain the value of using comparative genomics to learn about the properties of genomes. 2. Explain how functional genomics is used to learn about the functions of genomes. 3. Describe the relationships between the genome, the transcriptome, and the proteome.

Comparative genomics uses information from one genome to learn about a second genome. For example, the known function of a gene in one organism’s genome can be used to hypothesize about the function of a similar gene in a related organism’s genome. It turns out that 60% of the genes involved in triggering human cancer are conserved in the fruit fly genome. So, the functions of those genes can be analyzed in the fruit fly, which is significantly easier than analyzing them in humans. Comparative genomics also reveals information about the relatedness of organisms, about how different organisms perform similar biological functions, about what makes different species different, and even about the minimum number of genes it would take to build a functional cell—an engineering feat of interest to synthetic biologists discussed in section 18.6. Functional genomics is an extension of genomics that investigates the relationship between genotype and phenotype. The phenotype of an organism is determined by the pattern of expression of genes and by the interaction of the organism with the environment. For example, by comparing the gene expression pattern in a healthy cell with the gene expression pattern in a diseased cell, it is possible to learn which genes might be involved in the disease process.

Comparative genomics reveals conserved regions in genomes One of the striking lessons learned from the sequence of the human genome is how similar humans are to other organisms. More than half of the genes of Drosophila have human counterparts. Among mammals, the similarities are even greater. Humans have only 300 genes that have no counterpart in the mouse genome. The use of comparative genomics to ask evolutionary questions is also a field of great promise. The comparison of the many prokaryotic genomes already sequenced indicates a greater degree of lateral gene transfer than was previously suspected.

Comparative genomics based on synteny Comparative genomics allows for a large-scale approach to comparing genomes by taking advantage of synteny. Synteny refers to the conserved arrangements of segments of DNA in related genomes. Physical mapping techniques can be used to look for synteny in genomes that have not been sequenced. Comparisons with the sequenced, syntenous segment in another species can provide information about the unsequenced genome.

Rice Genome

Sugarcane Chromosome Segments

Genomic Alignment (Segment Rearrangement)

1 2 3 4 5 6

7 8 9 10 11 12

Corn Chromosome Segments

A B C D F G H

I

Wheat Chromosome Segments

R ice

S u g a rc a n e C orn

Whe at

1 2 3 4 5 6 7 8 9 10

1

2 3 4 5 6 7

Figure 18.10 Grain genomes are rearrangements of similar chromosome segments. Shades of the same color represent pieces of DNA that are conserved among the different species but have been rearranged. By splitting the individual chromosomes of major grass species into segments and rearranging the segments, researchers have found that the genome components of rice, sugarcane, corn, and wheat are highly conserved. This implies that the order of the segments in the ancestral grass genome has been rearranged by recombination as the grasses have evolved.

To illustrate this, consider rice and its grain relatives, corn, barley, and wheat. Only rice and corn genomes have been fully sequenced. Even though these plants diverged more than 50 million years ago (MYA), the chromosomes of rice, corn, wheat, and other grass crops show extensive synteny (figure 18.10). The information gleaned from the physical and genetic maps aids in sequencing projects for various cereal grains. Genome sequences have been, or are being, assembled for wheat, rice, barley, and sorghum. Comparisons between these reveal a set of 12,607 gene families that appear to represent the core genes for the cereal grains. These data will prove valuable in identifying genes associated with disease resistance, crop yield, and nutritional quality. One common feature of all of these genomes is that they contain a large amount of repetitive DNA, including transposable elements. The amount of the genome represented by transposable elements ranges from 60–80%. Recombination between dispersed repeated elements probably contributes to the degree of rearrangements visible at the level of the physical maps.

Functional genomics reveals gene function at the genome level To fully understand how a genome contributes to the structure and function of an organism, we need to characterize the products of the genome—the RNA molecules and the proteins. This information is essential to understanding cell biology, physiology, development, and evolution. Functional genomics allows connections to be drawn between the genotype of the organism and the phenotype of the organism. Functional genomics uses a range of high-throughput techniques to learn about the products produced from a genome, when those products are produced, and how they change during development or in response to the environment. Functional genomics can be broken into three separate, but related, categories: (1) the study of all the RNA molecules produced by a genome (the transcriptome); (2) the study of the proteins produced from the genome (the proteome); and (3) the study of the interactions and products of interactions made between proteins. We consider proteomics later chapter

18 Genomics 381

SCIENTIFIC THINKING Hypothesis: Flowers and leaves will express some of the same genes. Prediction: When mRNAs isolated from Arabidopsis flowers and from leaves are used as probes on an Arabidopsis genome microarray, the two different probe sets will hybridize to both common and unique sequences. Test: 2. DNA is printed onto a microscope slide.

1. Start with an Arabidopsis genome microarray. Unique, PCR-amplified Arabidopsis genome fragments (1, 2, 3, 4...) are contained in each well of a plate.

Robotic quill

Plate containing genome fragments

2

DNA microarray

Microscope slide 1

4

3 DNA 1

3. Isolate mRNA from flowers and leaves, convert to cDNA, and label with fluorescent labels. Samples of mRNA are obtained from two different tissues. Probes for each sample are prepared using a different fluorescent nucleotide for each sample. Flower-specific mRNA (sample 1)

2

3

4

4. Probe microarray with labeled cDNA. The two probes are mixed and hybridized with the microarray. Fluorescent signals on the microarray are analyzed. Probe 1 Mix Hybridize

Reverse transcriptase Fluorescent nucleotide

Probe 2 cDNA probe Leaf-specific mRNA (sample 2) Reverse transcriptase Different fluorescent nucleotide

Weak signal from probe 2 Similar signals from both probes Strong signal from probe 2

Strong signal from probe 1

Weak signal from probe 1

cDNA probe Result: Yellow spots represent sequences that hybridized to cDNA from both flowers and leaves. Red spots represent genes expressed only in flowers. Green spots represent genes expressed only in leaves. Conclusion: Some Arabidopsis genes are expressed in both flowers and leaves, but there are genes expressed in flowers but not leaves and leaves but not flowers. Further Experiments: How could you use microarrays to determine whether the genes expressed in both flowers and leaves are housekeeping genes or are unique to flowers and leaves?

Figure 18.11 Microarrays. in this section, but first we summarize several of the technologies used to study the transcriptome.

DNA microarrays DNA microarrays were created for the analysis of gene expression at the whole-genome level. The arrays allow questions to be asked about how gene expression patterns change during development, in 382 part III Genetic and Molecular Biology

response to environmental stimuli, and even during the development of disease. This kind of analysis requires accurate annotation of a genome and using this knowledge to construct an array containing all of the coding sequences (figure 18.11). To prepare a microarray, fragments of DNA are applied onto a glass or silicone slide by a robot at indexed locations to produce an array of DNA dots. Each DNA fragment

corresponds to specific gene sequences such that the entire array represents all possible coding transcripts, with unique locations in the array. The use of a microarray is shown in figure 18.11. Chips can be used in hybridization experiments with labeled mRNA from different sources. This gives a high-level view of genes that are active and inactive in a variety of different conditions or states. Arrays can also be prepared so that the spotted DNA contains variations of the DNA sequence so that only transcripts from genes with single-nucleotide polymorphisms are detected. Microarrays have some limitations, however. The sequence of a genome must be known to prepare a microarray with appropriate sequences. Also, if a microarray is being prepared to look for rare polymorphic transcripts in a sample, then knowledge of those polymorphisms is required for the array to be created. Because of some of these limitations, and given technological advances, other approaches have become popular in studying the transcriptome.

RNA sequencing (RNA-seq) High-throughput methods to sequence cellular RNAs are called RNA-seq. RNA-seq provides a snapshot of all of the mRNA in a sample, by directly sequencing cDNA. The sequencing is done using next-generation sequencing technologies. RNA-seq can be modified based on the experimental questions being asked. For example, RNA-seq can be used to identify intron-exon boundaries, map single-nucleotide polymorphisms to unsequenced genomes, and determine the contribution of different alleles to phenotype when alleles of a gene are present. RNA-seq suffers from the problem that in many cases it is not the RNA that produces a phenotype—rather, it is the protein. The presence of an RNA detected by RNA-seq does not take into account that the RNA may be posttranscriptionally regulated in a way that interferes with protein levels. Although analysis of the transcriptome of a cell provides a large amount of potentially important information, it can be critical to also consider the complete collection of proteins in a cell at a particular time. These studies comprise a field of functional genomics called proteomics.

Chromosome immunoprecipitation (ChIP-Seq) Antibodies can be used to not only recognize specific proteins, but also isolate them from a complex mixture by precipitation. This has been modified to find the genomic location for proteins bound to DNA. This has been primarily used to map the locations of bound transcription factors in a genome. This allows the analysis of actual protein binding, and not just the location of recognition sequences. Chromatin is isolated from cells, then antibodies are used to precipitate bound transcription factors. By sequencing the DNA bound in this complex, this can be related back to the genome sequence to map the location. The analysis by the ENCODE project described in section 18.4 used high-throughput variations on this kind of analysis to map genomic regions with a biological function (transcription factor binding).

Proteomics catalogs proteins encoded by the genome The proteome is much harder to analyze than either the genome or the transcriptome because proteins are harder to analyze than

nucleic acids. Proteins must fold properly to function; proteins are subject to posttranslational modifications that affect function; and a single gene can produce multiple proteins by alternative splicing (see chapter 15). All of this makes identifying the relationships between genomes, transcriptomes, and proteomes a difficult task. Many high-throughput techniques are being developed to allow identification of makeup of the proteome. The technique that has been the most useful to analyze the proteome is mass spectrometry (mass spec). Mass spec determines the charge-to-mass ratio of a molecule, which with small molecules allows for unambiguous identification. For many years, this was not used with proteins due to their size. However, new techniques combine purification and ionization to allow the use of mass spec to analyze complex mixtures of proteins (figure 18.12). One advantage of this technique is that it can reveal posttranslational modifications like phosphorylation. In interpreting data from this kind of analysis, it is important to recognize that the technique identifies relatively short peptides, and not complete proteins, requiring the use of computer-based analysis to identify cellular proteins. This has allowed an international consortium to identify proteins encoded by 90% of annotated human genes. Proteomics also uses the microarray technology described earlier in this section. Rather than DNA being spotted on the chip, antibodies recognizing specific proteins can be applied to the chip. If a mixture of proteins isolated from a sample is applied to an antibody chip, then the antibodies will bind to specific proteins in much the same way they do in the immunoassays described in chapter 17. Using this approach, specific proteins, known as biomarkers, can rapidly be identified in a complex sample. As well as being useful in a research setting, this technique has potential for disease diagnosis and screening.

Bioinformatics and proteomics Just like genome and transcriptome analysis, high-throughput proteomics analyses can produce huge amounts of data. The data are largely meaningless without approaches to study and interpret the data. At the interface between computer science and biology lies the discipline of bioinformatics. Bioinformatics is the application of computer programming, mathematics, and modeling to the analysis of large sets of biological data. The application of bioinformatics to proteomics allows the rapid identification of proteins discovered by high-throughput profiling experiments, the annotation of genomes using information from mass spectroscopy and protein chips, and the prediction of protein structure and function.

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Inquiry question How might you annotate a gene’s

function in a genome database if provided with the sequence of amino acids in a protein that was obtained from a proteomics experiment?

Predicting protein structure and function The use of new computer methods to quickly identify and characterize large numbers of proteins is a distinguishing feature between traditional protein biochemistry and proteomics. As with genomics, the challenge is one of scale. chapter

18 Genomics 383

Cells or tissue

Separate proteins

Protein mixture

Digestion into peptides

+

Peptide mixture

Separation of peptides by chromatography

Electrospray ionization

Ion-peptide

100

400

800 MS Spectrum

1200

m/z

Relative Abundance (%)

Relative Abundance (%)

Fragment peptide Peptide sequence Alanine Glycine Leucine

100

200

600 1000 MS/MS Spectrum

m/z

Figure 18.12 Mass spectrometry can be used to analyze proteins. Proteins can be isolated from cells or tissues and partially

separated using gels or biochemical techniques. Partially purified or individual proteins are digested into small peptides by proteases and ionized. The charged peptides can be separated by mass in a spectrometer. Computer algorithms can match the peptide fragments with predicted digestion patterns to tentatively identify proteins. Alternatively, individual peptides can be further fragmented and amino acid composition can be determined by mass spectrometry. (MS spectrum, abundance of an ion as a function of mass to charge ratio of the ion; MS/MS spectrum, abundance of an ion as a function of mass to charge ratio of the ion when the ions are the product of tandem ionization events; m/z, the mass of an ion divided by the charge of an ion, commonly referred to as the mass-to-charge ratio.)

Ideally, we would like to use a gene sequence to infer the structure and function of the encoded protein. At present, we can do this only by comparing a new protein sequence with the sequences of already identified proteins. Inferences about structure and function are sometimes possible due to the fact that similar sequences of amino acids will produce a similar structure. However, it is also true that similar structures can be produced from very different sequences of amino acids. In these cases, similar functions can be inferred only if structural information is available. Inference of function from structure is an even more challenging task. Proteins with similar sequences and structures often have similar functions, but there are many examples where this is not the case. There are many similar functions performed by proteins with significantly different structures. It is helpful if we have information about the evolutionary history of a protein. We can be more confident in our inferences about shared structures and functions if we are comparing proteins derived from a common ancestor that have not diverged in function. 384 part III Genetic and Molecular Biology

Until recently, it has been very difficult to predict the variety of ways in which chains of amino acids could fold into secondary and tertiary structures (an example of predicted protein structure is shown in figure 18.13). One type of computer algorithm attempts to predict protein structure by first looking for stretches of amino acids that have characteristics of a protein domain. Then, the algorithm takes short fragments from the protein domain and compares them to known folds in proteins whose structures are known. A computer program then uses these data to build a model of how all the fragments might be arranged to produce specific structures. When the same structure appears independently from different simulations, there is a higher probability that the structure is correct.

?

Inquiry question What is the relationship between genome, transcriptome, and proteome?

Genomics can be used to answer fundamental questions about biology. For example, we can better understand what makes one species fundamentally different from another and the role the genome plays in determining phenotype by understanding the functions of a genome, and the evolutionary relationships between different species’ genomes. With the application of the information we now have about genome structure and function come a number of social and ethical concerns.

Genome data were used to create the first “synthetic” cell

Figure 18.13 Computer-generated model of an enzyme. Searchable databases contain known protein structures,

including human aldose reductase, shown here. Secondary structural motifs are shown in different colors.  ©2015 David Goodsell & RCSB Protein Data Bank (www.rcsb.org). Molecule of the Month DOI 10.2210/rcsb_pdb/ mom_2013_10

Learning Outcomes Review 18.5

Comparisons of different genomes allow geneticists to infer structural, functional, and evolutionary relationships between genes and proteins, as well as relationships between species. Functional genomics provides tools to begin to understand the functions of the genes in a genome. DNA microarrays, SAGE, and RNA-seq can be used to learn about the transcriptome, but there are pros and cons to the different approaches. Proteomics involves analyses of the proteins produced by a genome under specific conditions. Although it is possible to make some predictions about protein structure and function, the analyses are complex and require powerful computers. ■■

Why is establishment of a species’ transcriptome an important step in studying its proteome?

18.6 Applications

of Genomics

Learning Outcome 1. Evaluate the ethical and social issues arising from the use of genomics data.

Life is characterized by a set of properties emerging from the assembly of atoms into molecules, molecules into macromolecules, and macromolecules into cellular structures. The pathways controlling the synthesis of those molecules and the processes leading to the formation of cellular structures are under the control of the genome. But what is the minimum genome size required to build an autonomously replicating cell? We can get some idea of the smallest minimum genome required to sustain life by finding the smallest genome in a living organism. The smallest viral genome is 5386 nt, but many scientists do not consider viruses to be alive as they are unable to replicate independently. The genome of the semiautonomous mitochondrion is about 16,600 nt long; however, the mitochondrion is unable to replicate independently of its cellular “host.” The smallest free-living bacterial genome currently known is that of Mycoplasma genitalium at about 500,000 nt. This implies that the minimum viable genome for a living cell is somewhere between 16,600 and 500,000 nt long. In 2010, scientists at the J. Craig Venter Institute announced that they had designed a Mycoplasma mycoides genome using a computer, chemically synthesized the DNA, assembled the DNA into a chromosome inside yeast cells, and transferred the chromosome into the bacterium Mycoplasma capricolum. The genome of the recipient bacterium was destroyed in the process and the new synthetic genome was able to control the growth of the new bacterial cell, named Mycoplasma mycoides JCVI-syn1.0 (figure  18.14). This approach may allow experimental verification of a minimal genome.

Genomics can help to identify and treat disease The genomics revolution has yielded millions of new genes to be investigated. The potential of genomics to improve human health is enormous. Mutations in a single gene can explain a limited number of hereditary diseases. However, genomics potentially allows us to look at any trait that is influenced by genetics. Although proteomics will likely lead to new pharmaceuticals, the immediate effect of genomics is being seen in diagnostics. Both improved technology and gene discovery are enhancing the diagnosis of genetic abnormalities. Diagnostics are also being used to identify individuals. For example, short tandem repeats (STRs), discovered through genomic research, were among the forensic diagnostic tools used to identify remains of victims of the September 11th, 2001, terrorist attack on the World Trade Center in New York City. Following the September 11th attacks in the United States, there was increased concern about biological weapons. Five people died and 17 more were infected with anthrax after envelopes containing spores were sent through the U.S. mail. Genome sequencing chapter

18 Genomics 385

Figure 18.14 Scanning electron micrograph of synthetic Mycoplasma mycoides JCVI-syn1.0 bacteria. ©Thomas Deerinck and Mark Ellisman, NCMIR, and John Glass, JCVI

allowed identification of the specific source of the bacteria used. A difference of only 10 bp between strains allowed the FBI to trace the source to a single vial of anthrax used in a vaccine research program at U.S. Army Medical Research Institute for Infectious Diseases. In the end, no one was formally charged by the FBI in these attacks, although a researcher under suspicion committed suicide, and another was exonerated. In the wake of this incident, the Centers for Disease Control and Prevention (CDC) has ranked bacteria and viruses that are likely targets for bioterrorism (table 18.2). Our knowledge of the human genome has also informed our search for the genetic basis of complex diseases. Studies referred to as genome-wide association studies (GWAS) look for association between genetic markers, like SNPs (see chapter 13), and traits or disease states. These studies are ongoing for traits like height—and for virtually every disease that has been shown to have a genetic component. Although a large number of genes have been identified this way, the effects of these genes do not account for the majority of known genetic variation.

Genomics raises social and ethical issues Genomics provides opportunities for understanding the relationship between our genotype and phenotype, as well as great potential for better diagnosing and treating some diseases. With these opportunities come social and ethical responsibilities regarding the use of the information. There must be careful consideration of the ethical and social consequences regarding ownership of genetic information and its uses, the potential misuses of the information, and even whether all genomics experiments should be performed.

Rebuilding the 1918 influenza virus In May of 2005, a series of papers describing the sequencing and reassembly of the 1918 Spanish influenza virus were published. The 1918 influenza virus killed between 20 and 50 million people. 386 part III Genetic and Molecular Biology

During the 1990s, several research groups, including some at the Centers for Disease Control and Prevention (CDC) in Atlanta and the Armed Forces Institute of Pathology in Washington, D.C., found fragments of the viral genome, sequenced them, and reconstructed the entire virus. The virus was found to have a similar mortality rate as the original virus when tested in other mammals such as mice. It has obviously not been tested in humans. These experiments raised serious concerns for some scientists, politicians, public interest groups, and the public in general. On the one hand, being able to study the genome of the virus, and the properties of the virus itself, may lead to more effective vaccines and help prevent future pandemics. Critics of these experiments claim that the vague potential benefits of recreating the virus are not worth the risk should an unfortunate or negligent scientist accidentally or deliberately release the virus. Many people also expressed concerns about the information being used by bioterrorists to create a weaponized form of the virus. After extensive reviews by the scientific community, careful scrutiny by the CDC, and oversight from the National Science Advisory Board for Biosecurity, it was decided that the benefits of making the work publicly available outweighed the risks. Of course, such decisions are subjective, but by being well informed about genomics, it is possible to make a more reasoned decision about such a controversial topic.

Owning and patenting genes You may think that you are the owner of the information in your genes, but until 2013 it appeared that someone else could own the rights to the sequences of your genes. In the 1990s, a company called Myriad Genetics patented two genes that were known to be involved in the development of some forms of breast cancer. By patenting the genes BRCA1 and BRCA2, the company secured exclusive rights to use the sequences to test for the presence of particular variants of the genes. This formed the basis of a test that provided information regarding predisposition to certain types of breast cancer. Given the increased risks of breast cancer in women carrying particular variants of BRCA1 and BRCA2, and the seriousness of a potential cancer diagnosis, many people believed that

TA B L E 1 8 . 2

High-Priority Pathogens for Genomic Research

Pathogen

Disease

Genome*

Variola major

Smallpox

Complete

Bacillus anthracis

Anthrax

Complete

Yersinia pestis

Plague

Complete

Clostridium botulinum

Botulism

Complete

Francisella tularensis

Tularemia

Complete

Filoviruses

Ebola and Marburg hemorrhagic fever

Both are complete

Arenaviruses

Lassa fever and Argentine hemorrhagic fever

Both are complete

* These viruses and bacteria have multiple strains. “Complete” indicates that at least one has been sequenced. For example, the Florida strain of anthrax was the first to be sequenced.

not being able to secure a second diagnostic opinion was an infringement of civil liberties. After a lengthy battle through federal courts driven largely by the American Civil Liberties Union (ACLU), a case against Myriad Genetics arrived at the Supreme Court of the United States. In a unanimous ruling, the Court ruled that because Myriad Genetics had not invented the genes, they could not patent the sequences of the genes. In their ruling, the Court noted that certain derived sequences—cDNA sequences, for example—and their uses are patentable because they have been created.

?

Inquiry question What arguments could be used to

support an application for the patent of a synthetic gene that increases the yield of lipids in algae used for biofuel production?

Learning Outcomes Review 18.6

Genome sequencing, annotation, and comparative and functional genomics led to the discovery of genes involved in disease. Some of those genes can be used to better diagnose disease or to provide more accurate prognoses. Genomics can also be used to learn about disease outbreaks and epidemiology. Genomic knowledge carries social and ethical responsibilities. By consultation with bioethicists, policymakers, and various interest groups, regulatory agencies can become better informed about experimentation and appropriate data use. ■■

Does the cell created by replacement of its genome with a sequence constructed by humans represent a new form of life?

Chapter Review ©William C. Ray, Director, Bioinformatics and Computational Biology Division, Biophysics Program, The Ohio State University

18.1 Mapping Genomes Genomics uses many approaches to analyze entire genomes. Genomics uses genetic and physical maps to assign genetic landmarks, called markers, to positions in the genome. Genetic maps provide relative distances between genetic markers. Linkage mapping determines the relative positions of genetic markers in a genome using genetic recombination. Physical maps provide absolute distances between genetic markers. Restriction maps, chromosome maps, and STS maps all assign markers to an actual location in the sequence of a genome (figures 18.1–18.3). Physical maps can be correlated with genetic maps. Physical and genetic maps can be correlated. Cloned genes can be placed on both genetic and physical maps.

18.2  Sequencing Genomes Dideoxy terminator sequencing remains important in genome sequencing. Dideoxy terminator sequencing relies on altered nucleotide chemistry to terminate DNA synthesis with a fluorescent nucleotide (figure 18.4). Next-generation sequencing uses massively parallel technologies to increase throughput. The cost and time to sequence a genome has reduced about 10,000fold since the start of the Human Genome Project. Next-generation sequencers analyze isolated DNA in thousands of simultaneous reactions to produce massive amounts of data (figure 18.5). Sequenced genome fragments are assembled into complete sequences. Shotgun sequencing and assembly methods recreate a genome sequence from very short pieces of slightly overlapping DNA. Clone-contig sequencing and assembly methods use larger pieces of DNA and a tiling approach to build larger overlapping fragments to assemble the whole genome (figure 18.6).

18.3  Genome Projects The Human Genome Project has sequenced and mapped most of the human genome. A competitive race to assemble the sequence of the human genome produced rapid advances in technology and early completion. The human genome contains significantly fewer genes than predicted (figure 18.7). Sequencing many human genomes reveals enormous variation. Genetic variation is divided into single-nucleotide variation and structural variation, which can cause copy number variation. Any random genome has variants not in the reference genome. The pattern of variation gives insight into human demography and migration. The Wheat Genome Project illustrates improvements in genome assembly. There have been four iterations of the wheat genome, each more complete. The most recent version used longer, error-prone reads combined with many shorter reads.

18.4  Genome Annotation and Databases Genome annotation assigns function to DNA sequences in genomes. Protein-coding genes are identified by open reading frames in DNA sequence. Comparing unknown genes to known ones can be used to infer gene function. The majority of the human genome does not encode protein. Protein-encoding DNA includes single-copy genes, segmental duplications, multigene families, and tandem clusters. Noncoding DNA in eukaryotes makes up about 99% of DNA. Approximately 45% of the human genome is composed of transposable elements (table 18.1). The ENCODE project seeks to identify all functional elements in the human genome. The ENCODE consortium predicts that anywhere from 20 to 80% of the human genome is functional. Critics of their approach question the utility of a definition of function that does not integrate evolutionary constraints on genome function (figure 18.9). chapter

18 Genomics 387

18.5  Comparative and Functional Genomics

18.6  Applications of Genomics

Comparative genomics reveals conserved regions in genomes. More than half of the genes of Drosophila have human counterparts. The different cereal genomes have large regions of synteny.

Genome data were used to create the first “synthetic” cell. A genome designed on a computer and chemically synthesized was assembled in yeast and added to a bacterium to create a new cell (figure 18.14).

Functional genomics reveals gene function at the genome level. Functional genomics uses high-throughput approaches to analyze gene function and gene products. DNA microarrays monitor the expression of all of the genes in a cell at once (figure 18.11). SAGE and RNA-seq study the transcriptome directly.

Genomics can help to identify and treat disease. Genomics expands the number of diseases we can identify that are influenced by genetics. This may lead to much more personalized medicine. Genomics raises social and ethical issues. Social groups and bioethicists are concerned about creating synthetic cells, and about reconstructing dangerous viruses. Individual genes cannot be patented, but synthetic products such as cDNA corresponding to the gene can be.

Proteomics catalogs proteins encoded by the genome. Proteomics characterizes all of the proteins produced by a cell. Proteomics is complicated by posttranslational modifications and alternative splicing (figures 18.12 and 18.13).

Visual Summary Genomics includes Mapping genomes

Sequencing genomes

allows

Genome projects such as

Genetic maps

Physical maps absolute distance

relative distance

Gene markers

original method Dideoxy sequencing

types

Restriction maps Chromosome maps Contig

faster method Next-gen sequencing

includes

Human genome project

assembled using

Open reading frames

includes

Genome analysis Comparative genomics Functional genomics

Class of DNA

Wheat genome project Human variation

Genome annotation

Proteomics Proteincoding DNA

Noncoding DNA

Applications

include

Clone-contig assembly

Medicine

Shotgun assembly

SSRs

STS maps

Transposable elements

microRNA genes

Structural DNA

Long, noncoding RNA

Pseudogenes

Review Questions ©William C. Ray, Director, Bioinformatics and Computational Biology Division, Biophysics Program, The Ohio State University

U N D E R S TA N D 1. A genetic map provides a. b. c. d.

the sequence of the DNA in a genome. the relative position of genes on chromosomes. the location of sites of restriction enzyme cleavage in a known sequence of DNA. the banding pattern of a chromosome.

388 part III Genetic and Molecular Biology

2. What is an STS? a. b. c. d.

A unique sequence within the DNA that can be used for mapping A repeated sequence within the DNA that can be used for mapping An upstream element that allows for mapping of the 3′ region of a gene A sequence of DNA that overlaps with other sequenced fragments in clone-contig genome assembly

3. Approximately how many genes are there in the human genome? a. b.

2500 10,000

c. d.

20,000 100,000

4. An open reading frame (ORF) is distinguished by the presence of a. b. c. d.

a stop codon. a start codon. a sequence of DNA long enough to encode a protein. All of the choices are correct.

5. What is a BLAST search? a. b. c. d.

A mechanism for aligning consensus regions during wholegenome sequencing A search for similar gene sequences from other species A method of screening a DNA library A method for identifying ORFs

6. Why is repetitive DNA a problem in shotgun sequencing and genome assembly? a. b. c. d.

The repetitive DNA cannot easily be sequenced. There is a lack of uniqueness between sequenced fragments. The repetitive DNA makes the genome too GC-rich. It becomes too expensive and too time-intensive.

7. What is a proteome? a. The collection of all genes encoding proteins b. The collection of all proteins encoded by the genome c. The collection of all proteins present in a cell d. The amino acid sequence of a protein 8. Why can the transcriptome not be used to predict the proteome with complete accuracy? a. It cannot be sequenced as the genome can be. b. The transcriptome is too dynamic to be used to make predictions. c. Not all genes are transcribed. d. Many transcripts are alternatively spliced to produce different proteins.

A P P LY 1. If genomics found that the same mutation was present in all cancer patients that failed to respond to a particular treatment, what might be concluded? a. b. c. d.

That the mutation was responsible for the failure to respond to treatment That the mutation would be a good diagnostic tool That children of those patients have an increased risk for the same cancer That the patients all have the same type of cancer

2. ENCODE defines function in a biochemical way. Which of the following genomics approaches could increase the accuracy of ENCODE’s estimate of genome functionality in humans? a. b. c. d.

Functional genomics Comparative genomics Genome sequencing and assembly Choices a, b, and c would increase the accuracy of their estimate.

3. If I synthesized a protein by chemically linking amino acids together and added a few extra amino acids that made the protein more stable and therefore better at treating a disease, what argument would support a patent for the protein? a.

The protein is beneficial to humans and so I should be paid for my discovery.

b. c. d.

The protein is synthetic and different from the natural form. The protein was synthesized chemically. The protein is expensive to make and a patent would help me offset my costs of production.

4. The genome of the frog Xenopus tropicalis is tetraploid. What challenges might this present for genome sequencing and assembly? a. b. c. d.

There may be duplicated pieces of genome that make genome assembly difficult. There may be LINEs that make sequencing difficult. Genetic and physical maps cannot be made. Both b and c are correct.

5. What information can be obtained from a DNA microarray? a. b. c. d.

The sequence of a particular gene The presence of genes within a specific tissue The pattern of gene expression Differences between genomes

6. Which of the following is true regarding microarray technology and cancer? a. b. c. d.

A DNA microarray can determine the type of cancer. A DNA microarray can measure the response of a cancer to therapy. A DNA microarray can be used to predict whether the cancer will move from one place to another in the body. All of the choices are correct.

7. Comparative proteomics involves comparing the proteomes from two different cells or tissues from different conditions. In some cancer cells the retinoblastoma protein often appears to have a slightly higher molecular mass than in noncancerous cells, even though the proteins have identical amino acid sequences. What might explain this difference? a. b. c. d.

Posttranslational modification Alternative splicing Association with different proteins Mutation of the gene encoding the protein

SYNTHESIZE 1. You are in the early stages of a genome-sequencing project. You have isolated a number of clones from a bacterial artificial chromosome (BAC) library and mapped the inserts in these clones using STSs. Use the STSs shown to align the clones into a contiguous sequence of the genome (a contig). STS 3

STS 4

STS 5

Clone A  ————————————— STS 2

STS 3

Clone B  —————————— STS 5

STS 6

Clone C  ———————————— STS 3

STS 4

Clone D  ———————— STS 1

STS 2

Clone E  ———————

2. Genomic research can be used to determine if an outbreak of an infectious disease is natural or “intentional.” Explain what a genomic researcher would be looking for in a suspected intentional outbreak of a disease like anthrax.

chapter

18 Genomics 389

19 CHAPTER

19

Cellular Mechanisms of Development Chapter Contents 19.1

The Process of Development

19.2

Cell Division

19.3

Cell Differentiation

19.4

Nuclear Reprogramming

19.5

Pattern Formation

19.6

Evolution of Pattern Formation

19.7

Morphogenesis

Science Photo Library/Steve Gschmeissner/Getty Images

Introduction

Visual Outline Development Cell division

Differentiation

preceded by

from

Pattern formation

Morphogenesis involves

determines

Regulation

Body plan

Science Source

Determination due to

of

Stem cells

Cell division

undergo Cytoplasmic determinants Cell–cell interactions

Epigenetic changes

Anterior– posterior axis

Dorsal– ventral axis

Cell shape

Cell migration

Drosophila HOM Chromosomes Drosophila HOM genes

Cell death FGF signaling

Anterior

Fruit fly embryo

Posterior 32-Cell Stage

64-Cell Stage

Fruit fly

Extracellular matrix

Recent work with different kinds of stem cells, like those pictured, has captured the hopes and imagination of the public. For thousands of years, humans have wondered how organisms arise, grow, change, and mature. We are now in an era when long-standing questions may be answered, and new possibilities for regenerative medicine seem on the horizon. We have explored gene expression from the perspective of individual cells, examining the diverse mechanisms cells employ to control the transcription of particular genes. Now we broaden our perspective and look at the unique challenge posed by the development of a single cell, the fertilized egg, into a multicellular organism. In the course of this developmental journey, a pattern of gene expression takes place that causes particular lines of cells to proceed along different paths, spinning an incredibly complex web of cause

and effect. Yet, for all its complexity, this developmental program works with impressive precision. In this chapter, we explore the mechanisms of development at the cellular and molecular level.

19.2 Cell

Division

Learning Outcomes

19.1 The Process of Development

1. Characterize the role of cell division in early development. 2. Describe the use of C. elegans to track cell lineages. 3. Distinguish differences in cell division between animals and plants.

Learning Outcome 1. Describe the different processes involved in development.

Development can be defined as the process of systematic, genedirected changes through which an organism forms the successive stages of its life cycle. Development is a continuum, and explorations of development can be focused on any point along this continuum. The study of development plays a central role in unifying the understanding of both the similarities and the diversity of life on Earth. We can divide the overall process of development into four subprocesses: ■■

■■

■■

■■

Cell division. A developing plant or animal begins as a fertilized egg, or zygote, that must undergo cell division to produce the new individual. In all cases early development involves extensive cell division, but in many cases it does not include much growth as the egg cell itself is quite large. Differentiation. As cells divide, orchestrated changes in gene expression result in differences between cells that ultimately result in cell specialization. In differentiated cells, certain genes are expressed at particular times, but other genes may not be expressed at all. Pattern formation. Cells in a developing embryo must become oriented to the body plan of the organism the embryo will become. Pattern formation involves cells’ abilities to detect positional information that guides their ultimate fate. Morphogenesis. As development proceeds, the form of the body—its organs and anatomical features—is generated. Morphogenesis may involve cell death, cell division, cell migration, changes in cell shape, and differentiation.

Despite the overt differences between groups of plants and animals, most multicellular organisms develop according to molecular mechanisms that are fundamentally very similar. This observation suggests that these mechanisms evolved very early in the history of multicellular life. a.

0.8 mm

When a frog tadpole hatches out of its protective coats, it is roughly the same overall mass as the fertilized egg from which it came. Instead of being made up of just one cell, however, the tadpole consists of about a million cells, which are organized into tissues and organs with different functions. Thus, the very first process that must occur during embryogenesis is cell division. Immediately following fertilization, the diploid zygote undergoes a period of rapid mitotic divisions that ultimately result in an early embryo comprised of dozens to thousands of diploid cells.

Development begins with cell division In animal embryos, the timing and number of these divisions are species-specific, and this period of rapid cell division following fertilization is called cleavage. During cleavage, the enormous mass of the zygote is subdivided into a larger and larger number of smaller and smaller cells, called blastomeres (figure 19.1). Hence, cleavage is not accompanied by any increase in the overall size of the embryo. The G1 and G2 phases of the cell cycle, during which a cell increases its mass and size, are extremely shortened or eliminated during cleavage (figure 19.2). This is controlled by the mechanisms we examined in chapter 10, with cyclins and cyclindependent kinases (Cdks). These molecules exert control over checkpoints in the cycle of mitosis. Because of the absence of the two gap/growth phases, the rapid rate of mitotic divisions during cleavage are the fastest

Figure 19.1 Cleavage divisions in a frog embryo. a. The first cleavage division divides the egg into two large blastomeres. b. After two more divisions, four small blastomeres sit on top of four large blastomeres, each of which continues to divide to produce (c) a compact mass of cells. Science Source/Science Source

b.

0.8 mm

chapter

c.

0.8 mm

19 Cellular Mechanisms of Development 391

Cell Cycle of Early Frog Blastomere

Adult Cell Cycle Mitosis Active

Cdk / G2 cyclin

M

C

G2

Active

G1

Cdk / G1 cyclin

Cdk / cyclin

Active

Cyclin Synthesis

S DNA Synthesis

S G2

interphase

M

mitosis

C

cytokinesis

a.

Cyclin Degradation

M

Active

G1

Cdk / S cyclin

Mitosis

C

Inactive S

Cdk

DNA Synthesis

b.

Figure 19.2 Cell cycle of adult cell and embryonic cell. In contrast to the cell cycle of adult somatic cells (a), the dividing cells of early frog embryos lack G1 and G2 stages (b), enabling the cleavage stage nuclei to rapidly cycle between DNA synthesis and mitosis. Large stores of cyclin mRNA are present in the unfertilized egg. Periodic degradation of cyclin proteins correlates with exiting from mitosis. Cyclin degradation and Cdk inactivation allow the cell to complete mitosis and initiate the next round of DNA synthesis. divisions in the lifetime of any animal. For example, zebrafish blastomeres divide once every 15 minutes during cleavage, to create an embryo with a thousand cells in just under 3 hr! After 12 cycles, this will increase to 60 min. As a comparison, cycling adult human intestinal epithelial cells divide on average only once every 19 hr. A comparison of the different patterns of cleavage can be found in chapter 52. When external sources of nutrients become available—for example, during larval feeding stages or after implantation of mammalian embryos—daughter cells can increase in size following cytokinesis, and an overall increase in the size of the organism occurs as more cells are produced.

Every cell division is known in the development of C. elegans One of the most completely described models of development is the tiny nematode Caenorhabditis elegans. Only about 1 mm long, the adult worm consists of 959 somatic cells. Because C. elegans is transparent, individual cells can be followed as they divide. By observing them, researchers have learned how each of the cells that make up the adult worm is derived from the fertilized egg. As shown on the lineage map in figure 19.3a, the egg divides into two cells, and these daughter cells continue to divide. Each horizontal line on the map represents one round of cell division. The length of each vertical line represents the time between cell divisions, and the end of each vertical line represents one fully differentiated cell. In figure 19.3b, the major organs of the worm are color-coded to match the colors of the corresponding groups of cells on the lineage map. Some of these differentiated cells, such as some cells that generate the worm’s external cuticle, are “born” after only 8 rounds of cell division; other cuticle cells require as many as 14 rounds. The cells that make up the worm’s pharynx, or feeding organ, are born after 9 to 11 rounds of division, whereas cells in the gonads require up to 17 divisions. 392 part III Genetic and Molecular Biology

Exactly 302 nerve cells are destined for the worm’s nervous system. Exactly 131 cells are programmed to die, mostly within minutes of their “birth.” The fate of each cell is the same in every C. elegans individual, except for the cells that will become eggs and sperm.

Plant growth occurs in specific areas called meristems A major difference between animals and plants is that most animals are mobile, at least in some phase of their life cycles, and therefore they can move away from unfavorable circumstances. Plants, in contrast, are anchored in position and must simply endure whatever environment they experience. Plants compensate for this restriction by allowing development to accommodate local circumstances. Instead of creating a body in which every part is specified to have a fixed size and location, a plant assembles its body throughout its life span from a few types of modules, such as leaves, roots, branch nodes, and flowers. Each module has a rigidly controlled structure and organization, but how the modules are utilized is quite flexible—they can be adjusted to environmental conditions. Plants develop by building their bodies outward, creating new parts from groups of stem cells that are contained in structures called meristems. As meristematic stem cells continually divide, they produce cells that can differentiate into the tissues of the plant. This simple scheme indicates a need to control the process of cell division. We know that cell-cycle control genes are present in both yeast (fungi) and animal cells, implying that these are a eukaryotic innovation—and in fact, the plant cell cycle is regulated by the same mechanisms, namely through cyclins and cyclin-dependent kinases. In one experiment, overexpression of a Cdk inhibitor in transgenic Arabidopsis thaliana plants resulted in strong inhibition of cell division in leaf meristems, leading to significant changes in leaf size and shape.

Nematode Lineage Map Egg Egg and sperm line Nervous system

Pharynx

Development in C. elegans has been mapped out such that the fate of each cell from the single egg cell has been determined. a. The lineage map shows the number of cell divisions from the egg, and the color coding links their placement in (b) the adult organism.

Intestine

Cuticle-making cells

Vulva

Figure 19.3  Studying embryonic cell division and development in the nematode. 

Gonad

a. Adult Nematode Gonad

Gonad Sperm

Egg Vulva

Intestine Cuticle

Nervous system Pharynx

b.

Learning Outcomes Review 19.2

In animal embryos, a series of rapid cell divisions that skip the G1 and G2 phases convert the fertilized egg into many cells with no change in size. In the nematode C. elegans, every cell division leading to the adult form is known, and this pattern is invariant, allowing biologists to trace development in a cell-by-cell fashion. In plants, growth is restricted to specific areas called meristems, where undifferentiated stem cells are retained. ■■

How are early cell divisions in an embryo different from those in an adult organism?

19.3

Cell Differentiation

Learning Outcomes 1. Describe the progressive nature of determination. 2. Illustrate with examples how cells become committed to developmental pathways. 3. Differentiate between the different types of stem cells.

In chapter 16, we examined the mechanisms that control eukaryotic gene expression. These processes are critical for the development of multicellular organisms, which have many different tissues and organs. During development, cells become different from one another because of the differential expression of subsets of genes— both at different times, and in different locations of the developing embryo. We now examine this process in more detail.

Cells become determined prior to differentiation A human body contains around 300 types of differentiated cells. Different cells can be distinguished based on the particular proteins they synthesize, their morphologies, and their specific functions. The total number and type of human cells is being cataloged by a project called the Human Cell Atlas. In this effort, cells are differentiated primarily by patterns of gene expression, which is revealing a large diversity of subtypes of known cell types. Before overt differentiation takes place, cells make a molecular decision to become a particular cell type. This process of cell determination commits a cell to a particular developmental pathway.

Tracking determination Determination is often not visible in the cell and can only be detected experimentally. The standard experiment to test whether­ chapter

19 Cellular Mechanisms of Development 393

a cell or group of cells is determined is to move the Not Determined Determined Normal (early development) (later development) donor cell(s) to a different location in a host (recipient) embryo. If the cells of the transplant develop into the same type of cell that they would have if left unNo donor Donor disturbed, then they are judged to be already determined (figure 19.4). Tail cells are Tail cells are transplanted transplanted The time course of determination can be asto head to head sessed by transplantation experiments. For example, a cell in the prospective brain region of an Recipient amphibian embryo at the early gastrula stage Before Overt (see chapter 52) has not yet been determined; if Differentiation Tail Head transplanted elsewhere in the embryo, it will develop according to the site of transplant. By the late gastrula stage, however, additional cell interactions have occurred, determination has taken place, and Recipient After Overt the cell will develop as neural tissue no matter Differentiation where it is transplanted. Tail cells develop Tail cells develop Determination often takes place in stages, with a into head cells in head into tail cells in head cell first becoming partially committed, acquiring positional labels that reflect its location in the embryo. These labels can have a great influence on how the pat- Figure 19.4 The standard test for determination. The gray ovals represent tern of the body subsequently develops. In a chicken embryos at early stages of development. The cells to the right normally develop into embryo, tissue at the base of the leg bud normally gives head structures, whereas the cells to the left usually form tail structures. If prospective rise to the thigh. If this tissue is transplanted to the tip tail cells from an early embryo are transplanted to the opposite end of a host embryo, of the identical-looking wing bud, which would nor- they develop according to their new position into head structures. These cells are not mally give rise to the wing tip, the transplanted tissue determined. At later stages of development, the tail cells are determined since they now will develop into a toe rather than a thigh. The tissue develop into tail structures after transplantation into the opposite end of a host embryo! has already been determined as leg, but it is not yet Determination can be due committed to being a particular part of the leg. Therefore, it can be influenced by the positional signaling at the tip of the to cytoplasmic determinants wing bud to form a tip (but in this case, a tip of leg). Many invertebrate embryos provide good visual examples of cell determination through the differential inheritance of cytoplasmic The molecular basis of determination determinants. Tunicates are marine invertebrates (see chapter 34), and most adults have simple, saclike bodies that are attached to the Cells initiate developmental changes by using transcription factors underlying substratum. Tunicates are placed in the phylum Chorto change patterns of gene expression. When genes encoding these data, however, due to the characteristics of their swimming, tadtranscription factors are activated, one of their effects is to reinforce pole-like larval stage, which has a dorsal nerve cord and notochord their own activation. This reinforcement makes the developmental (figure 19.5a). The muscles that move the tail develop on either switch deterministic, initiating a chain of events that leads down a side of the notochord. particular developmental pathway. In many tunicate species, yellow-colored pigment granCells in which a set of regulatory genes have been activated ules become asymmetrically localized in the egg following fermay not actually undergo differentiation until some time later, when tilization and subsequently segregate to the tail muscle cell other factors interact with the regulatory protein and cause it to progenitors during cleavage (figure 19.5b). When these pigment activate still other genes. Nevertheless, once the initial “switch” is granules are shifted experimentally into other cells that normally thrown, the cell is fully committed to its future developmental path. do not develop into muscle, their fate is changed and they Cells become committed to follow a particular developmental become muscle cells. Thus, the molecules that flip the switch pathway in one of two ways: for muscle development appear to be associated with the pigment 1. via the differential inheritance of cytoplasmic granules. determinants, which are maternally produced and The next step is to determine the identity of the molecules deposited into the egg during oogenesis; or involved. Experiments indicate that the female parent provides the 2. via cell–cell interactions. egg with mRNA encoded by the macho-1 gene. The elimination of macho-1 function leads to a loss of tail muscle in the tadpole, and The first situation can be likened to a person’s social status the misexpression of macho-1 mRNA leads to the formation of being determined by who his or her parents are and what he or she additional (ectopic) muscle cells from nonmuscle lineage cells. has inherited. In the second situation, the person’s social standing The macho-1 gene product has been shown to be a transcription is determined by interactions with his or her neighbors. Clearly factor that can activate the expression of several muscle-specific both can be powerful factors in the development and maturation of genes. that individual. 394 part III Genetic and Molecular Biology

MEIOSIS

Sperm (haploid) n

Egg (haploid) n FER

Adult tunicate (diploid) 2n

IZ TIL

IO AT

N

n 2n

Pigment granules Embryo (diploid) 2n

ME TAM ORP HOSIS

Larva (diploid) 2n

a.

b.

Figure 19.5 Muscle determinants in tunicates. a. The life cycle of a solitary tunicate. Muscle cells that move the tail of the swimming tadpole are arranged on either side of the notochord and nerve cord. The tail is lost during metamorphosis into the sedentary adult. b. The egg of the tunicate Styela contains bright yellow-colored pigment granules. These become asymmetrically localized in the egg following fertilization, and cells that inherit the yellow-colored granules during cleavage will become the larval muscle cells. Embryos at the 2-cell, 4-cell, 8-cell, and 64-cell stages are shown. The tadpole tail will grow out from the lower region of the embryo in the bottom panel. (b) ©J.Richard Whittaker, used by

permission

Induction can lead to cell differentiation In chapter 9, we examined a variety of ways by which cells communicate with one another. We can demonstrate the importance of cell–cell interactions in development by separating the cells of an early frog embryo and allowing them to develop independently. Under these conditions, blastomeres from one pole of the embryo (the “animal pole”) develop features of ectoderm, and blastomeres from the opposite pole of the embryo (the “vegetal pole”) develop features of endoderm. None of the two separated groups of cells ever develop features characteristic of mesoderm, the third main cell type. If animal-pole cells and vegetalpole cells are placed next to each other, however, some of the animal-pole cells develop as mesoderm. The interaction between the two cell types triggers a switch in the developmental path of these cells. This change in cell fate due to interaction with an adjacent cell is called induction. Signaling molecules act to alter gene expression in the target cells, in this case, some of the animal-pole cells. Another example of inductive cell interactions is the formation of the notochord and mesenchyme, a specific tissue,

in tunicate embryos. Muscle, notochord, and mesenchyme all arise from mesodermal cells that form at the vegetal margin of the 32-cell-stage embryo. These prospective mesodermal cells receive signals from the underlying endodermal precursor cells that lead to the formation of notochord and mesenchyme (figure 19.6). The chemical signal is a member of the fibroblast growth factor (FGF) family of signaling molecules. It induces the over­ lying marginal zone cells to differentiate into either notochord (anterior) or mesenchyme (posterior). The FGF receptor on the marginal zone cells is a receptor tyrosine kinase that transmits signals via a MAP kinase cascade. This activates a transcription factor that turns on the expression of genes required for differentiation (figure 19.7). This example is also a case of two cells responding differently to the same signal. The presence or absence of the ­macho-1 muscle determinant controls this difference in cell fate. In the presence of macho-1, cells differentiate into mesenchyme; in its absence, cells differentiate into notochord. Thus, the combination of macho-1 and FGF signaling leads to four different cell types (figure 19.7). chapter

19 Cellular Mechanisms of Development 395

Sagittal section 1 2

Longitudinal section

Stem cells can divide and produce cells that differentiate

1

Anterior

Anterior

Posterior

Dorsal nerve cord (NC)

Notochord (Not)

Ventral endoderm (En) Longitudinal section

2

Mesenchymal cells (Mes)

Posterior Tail muscle cells (Mus)

a. FGF signaling

Anterior

Posterior 32-cell stage

b.

64-cell stage

c.

Figure 19.6 Inductive interactions contribute to cell fate specification in tunicate embryos. a. Internal structures of a tunicate larva. To the left is a sagittal section through the larva with dotted lines indicating two longitudinal sections. Section 1, through the midline of a tadpole, shows the dorsal nerve cord (NC), the underlying notochord (Not), and the ventral endoderm cells (En). Section 2, a more lateral section, shows the mesenchymal cells (Mes) and the tail muscle cells (Mus). b. View of the 32-cell stage looking up at the endoderm precursor cells. Fibroblast growth factor (FGF) secreted by these cells is indicated with light-green arrows. Only the surfaces of the marginal cells that directly border the endoderm precursor cells bind FGF signal molecules. Note that the posterior vegetal blastomeres also contain the macho-1 determinants (red and white stripes). c. Cell fates have been fixed by the 64-cell stage. Colors are as in (a). Cells on the anterior margin of the endoderm precursor cells become notochord and nerve cord, respectively, whereas cells that border the posterior margin of the endoderm cells become mesenchyme and muscle cells, respectively. 396 part III Genetic and Molecular Biology

It is important, both during development and even in the adult animal, to have cells set aside that can divide but are not determined for only a single cell fate. The name of cells that are capable of continued division but that can also give rise to differentiated cells is stem cells. These cells can be characterized based on the degree to which they have become determined. At one extreme, we call a cell that can give rise to any tissue in an organism totipotent. In mammals, the only cells that can give rise to both the embryo and the extraembryonic membranes are the zygote and early blastomeres from the first few cell divisions. Cells that can give rise to all of the cells in the organism’s body are called ­pluripotent. A stem cell that can give rise to a limited number of cell types, such as the cells that give rise to the different blood cell types, are called multipotent. Then at the other extreme, unipotent stem cells give rise to only a single cell type, such as the cells that give rise to sperm cells in males.

Embryonic stem cells are pluripotent cells derived from embryos Embryonic stem cells (ES cells) are a form of pluripotent stem cells isolated in the laboratory. These cells are made from mammalian embryos at the blastocyst stage of development. The blastocyst consists of an outer ball of cells, the trophectoderm, which will become extraembryonic structures such as the placenta, and the inner cell mass that will go on to form the embryo (see chapter 52 for details). Embryonic stem cells are essentially cells from the inner cell mass grown in culture (figure 19.8). In mice, these cells have been shown to be able to develop into any type of cell in the tissues of the adult. However, these cells cannot give rise to the extraembryonic tissues that arise during development, so they are pluripotent, but not totipotent. Once these cells were found in mice, it was only a matter of time before human ES cells were derived as well. In 1998, the first human ES cells (hES cells) were isolated and grown in culture. Although there are differences between human and mouse ES cells, there are also substantial similarities. These embryonic stem cells hold great promise for regenerative medicine based on their potential to produce any cell type as described here. These cells have also been the source of much controversy and ethical discussion due to their embryonic origin.

Differentiation in culture In addition to their possible therapeutic uses, ES cells offer a way to study the differentiation process in culture. The manipulation of these cells by additions to the culture media will allow us to tease out the factors involved in differentiation at the level of the actual cell undergoing the process. Early attempts at assessing differentiation in culture were plagued by the culture conditions. The medium in early experiments contained fetal calf serum (common in tissue culture), which is ill-defined, and varies from lot to lot. More recently, more defined culture conditions have been found that allow greater reproducibility in controlling differentiation in culture. Using more defined media, ES cells have been used to recapitulate in culture the early events in mouse development.

First Step

Macho-1 inherited?

Second Step

Cell Types

FGF Signal received?

Yes

FGF Signal received?

No

Yes No

Mesenchyme

Yes No

Notochord

Muscle Nerve cord

a. No FGF

FGF

FGF Receptor

FGF Receptor

Cell membrane

Cell membrane Ras/MAPK Pathway

Ras/MAPK Pathway

T-Ets

Macho-1

T-Ets

No FGF

FGF FGF Receptor

FGF Receptor

Cell membrane

Cell membrane Ras/MAPK Pathway

Ras/MAPK Pathway

T-Ets

Macho-1

T-Ets

No Macho-1

No Macho-1

P

P Suppression of muscle genes and activation of mesenchyme genes

Mesenchyme Precursor Cells

Transcription of muscle genes

Transcription of notochord genes

Muscle Precursor Cells

Suppression of notochord genes and activation of nerve cord genes

Notochord Precursor Cells

Nerve cord Precursor Cells

b.

Figure 19.7 Model for cell fate specification by Macho-1 muscle determinant and FGF signaling. a. Two-step model of cell fate specification in vegetal marginal cells of the tunicate embryo. The first step is inheritance (or not) of muscle macho-1 mRNA. The second step is FGF signaling from the underlying endoderm precursor cells. b. Posterior vegetal margin cells inherit macho-1 mRNA. Signaling by FGF activates a Ras/ MAP kinase pathway that produces the transcription factor T-Ets. Macho-1 protein and T-Ets suppress muscle-specific genes and turn on mesenchymespecific genes (green cells). In cells with Macho-1 that do not receive the FGF signal, Macho-1 alone turns on muscle-specific cells (yellow cells). Anterior vegetal margin cells do not inherit macho-1 mRNA. If these cells receive the FGF signal, T-Ets turns on notochord-specific genes (purple cells). In cells that lack Macho-1 and FGF, notochord-specific genes are suppressed and nerve cord–specific genes are activated (gray cells).

Data analysis What type of cells would develop if you injected embryos with a reagent that blocked the FGF receptor, thus preventing its signaling? What about with a reagent that turned on the FGF receptor, thereby causing it to be always on?

Thus mouse ES cells can be used to first give rise to ectoderm, endoderm, and mesoderm; then these three cell types will give rise to the different cells each germ layer is determined to become. This work is in early stages but is tremendously exciting as it offers the promise of understanding the molecular cues that are involved in the stepwise determination of different cell types. In humans, ES cells have been used to give rise to a variety of cell types in culture. For example, human ES cells have been shown to give rise to different kinds of blood cells in culture. Work is under way to produce hematopoietic stem cells in culture, which could be used to replace such cells in patients with diseases that affect blood cells. Human ES cells have also been used to produce cardiomyocytes in culture. These cells could be used to replace damaged heart tissue after heart attacks.

Learning Outcomes Review 19.3

Cell differentiation is preceded by determination, where the cell becomes committed to a developmental pathway, but has not yet differentiated. Differential inheritance of cytoplasmic factors can cause determination and differentiation, as can interactions between neighboring cells (induction). Inductive changes are mediated by signaling molecules that trigger transduction pathways. Stem cells are able to divide indefinitely, and they can give rise to differentiated cells. Embryonic stem cells are pluripotent cells that can give rise to all adult structures. ■■

How could you distinguish whether a cell becomes determined by induction or because of cytoplasmic factors?

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19 Cellular Mechanisms of Development 397

Once sperm cell and egg cell have joined, cell cleavage produces a blastocyst. The inner cell mass of the blastocyst develops into the human embryo.

Egg

Sperm

Embryonic stem cell culture

Inner cell mass

Blastocyst

Embryo

Embryonic stem cells (ES cells) are isolated from the inner cell mass

a.

500 μm

b.

Figure 19.8 Isolation of embryonic stem cells. a. Early cell divisions lead to the blastocyst stage that consists of an outer layer and an

inner cell mass, which will go on to form the embryo. Embryonic stem cells (ES cells) can be isolated from this stage by disrupting the embryo and plating the cells. Stem cells removed from a six-day blastocyst can be established in culture and maintained indefinitely in an undifferentiated state. b. Human embryonic stem cells. This mass in the photograph is a colony of undifferentiated human embryonic stem cells being studied in the developmental biologist James Thomson’s research lab at the University of Wisconsin–Madison.

(b) Deco Images II/Alamy Stock Photo

19.4

Nuclear Reprogramming

Learning Outcomes 1. Contrast different methods of nuclear reprogramming. 2. Differentiate between reproductive and therapeutic cloning.

The process of going from a single-celled zygote to a complex multicellular animal does not involve any irreversible changes to the DNA. This implies that the changes that underlie determination and differentiation are epigenetic processes. An epigenetic change does not change the DNA, but is stable through cell division. These types of changes include DNA methylation and the modification of histone proteins, which can affect chromatin structure and gene expression (see chapter 16). During the normal

Figure 19.9 Proof that determination in animals is reversible. Scientists combined a nucleus from an adult mammary cell with an

enucleated egg cell to successfully clone a sheep, named Dolly, who grew to be a normal adult and bore healthy offspring. This experiment, the first successful cloning of an adult animal, shows that a differentiated adult cell can be used to drive all of development. (right) Paul Clements, File/AP Images

Data analysis The sheep used for the donor nucleus had a different pattern of pigmentation than the donor egg. Why is this important, and which animal should Dolly resemble? Preparation

Cell Fusion

Mammary cell is extracted and grown in nutrient-deficient solution that arrests the cell cycle. Nucleus containing source DNA

Egg cell is extracted.

398 part III Genetic and Molecular Biology

Nucleus is removed from egg cell with a micropipette.

Mammary cell is inserted inside covering of egg cell.

Electric shock fuses cell membranes and triggers cell division.

Cell Division

course of development, this epigenetic program changes from differentiated adult cells, to germ cells, to the zygote itself.

Reversal of determination has allowed cloning The experimental reprogramming of nuclei has a long and interesting history. This is of interest to understand the basic process, and also raises the prospect of creating patient-specific populations of specific cell types to replace cells lost to disease or trauma. This has led to a fascinating path with many twists and turns that continues today. Experiments carried out in the 1950s showed that single cells from fully differentiated tissue of an adult plant could develop into entire, mature plants. The cells of an early cleavage stage mammalian embryo are also totipotent. When mammalian embryos naturally split in two, identical twins result. If individual blastomeres are separated from one another, any one of them can produce a completely normal individual. In fact, this type of procedure has been used to produce sets of four or eight identical offspring in the commercial breeding of particularly valuable lines of cattle.

differentiated cell undergoes ­epigenetic changes that must be reversed to allow the nucleus to direct development. The early work on amphibians showed that tadpoles’ intestinal cell nuclei could be reprogrammed to produce viable adult frogs. Not only can these animals be considered clones, but they also show that tadpole nuclei can be completely reprogrammed. However, nuclei from adult differentiated cells could be reprogrammed to produce only tadpoles, but not viable, fertile adults. Thus this work showed that adult nuclei have remarkable developmental potential, but cannot be reprogrammed to be totipotent.

Early research in mammals Given the work done in amphibians, much effort was put into nuclear transfer in mammals, primarily mice and cattle. Not only did this not result in reproducible production of cloned animals, but this work led to the discovery of imprinting through the production of embryos with only maternal or paternal input (see chapter 13 for more information on imprinting). These embryos never developed, and showed different kinds of defects depending on whether the maternal or paternal genome was the sole contributor.

Successful nuclear transplant in mammals

Early research in amphibians An early question in developmental biology was whether the production of differentiated cells during development involved irreversible changes to cells. Experiments carried out in the 1950s by Robert Briggs and Thomas King, and by John Gurdon in the 1960s and 1970s, showed nuclei could be transplanted between cells. Using very fine pipettes (hollow glass tubes), these researchers sucked the nucleus out of a frog or toad egg and replaced the egg nucleus with a nucleus sucked out of a body cell taken from another individual. The conclusions from these experiments are somewhat contradictory. On the one hand, cells do not appear to undergo any truly irreversible changes, such as loss of genes. On the other hand, the more differentiated the cell type, the less successful the nucleus in directing development when transplanted. This led to the concept of nuclear reprogramming—that is, a nucleus from a

Development

Implantation

Birth of Clone

Embryo begins to develop in vitro.

Embryo is implanted into surrogate mother.

After a five-month pregnancy, a lamb genetically identical to the sheep from which the mammary cell was extracted is born.

These results stood until a sheep was cloned using the nucleus from a cell of an early embryo in 1984. The key to this success was in picking a donor cell very early in development. This exciting result was soon replicated by others in a host of other organisms, including pigs and monkeys. Only early embryo cells seemed to work, however. Geneticists at the Roslin Institute in Scotland reasoned that the egg and donated nucleus would need to be at the same stage of the cell cycle for successful development. To test this idea, they performed the following procedure (figure 19.9): 1. They removed differentiated mammary cells from the udder of a six-year-old sheep. The cells were grown in tissue culture, and then the concentration of serum nutrients was substantially reduced for five days, causing them to pause at the beginning of the cell cycle.

Growth to Adulthood

Embryo

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19 Cellular Mechanisms of Development 399

2. In parallel preparation, eggs obtained from a ewe were enucleated. 3. Mammary cells and egg cells were surgically combined in a process called somatic cell nuclear transfer (SCNT) in January of 1996. Mammary cells and eggs were fused to introduce the mammary nucleus into an egg. 4. Twenty-nine of 277 fused couplets developed into embryos, which were then placed into the reproductive tracts of surrogate mothers. 5. A little over five months later, on July 5, 1996, one sheep gave birth to a lamb named Dolly, the first clone generated from a fully differentiated animal cell.

Oocyte

Somatic cells

Nuclear Transfer

Somatic cells Fusion

Blastocyst

Dolly matured into an adult ewe, and she was able to reproduce the old-fashioned way, producing six lambs. Thus, Dolly­ established beyond all dispute that determination in animals is reversible—that with the right techniques, the nucleus of a fully differentiated cell can be reprogrammed to be totipotent.

ES cells Defined Factors

Culture Pluripotent stem cells

Reproductive cloning has inherent problems The term reproductive cloning refers to the process just described, in which scientists use SCNT to create an animal that is genetically identical to another animal. Since the announcement of Dolly’s birth in 1997, scientists have successfully cloned one or more cats, dogs, rabbits, rats, mice, cattle, goats, horses, deer, oxen, pigs, and mules. All of these procedures used some form of adult cell.

Low success rate and age-associated diseases The efficiency in all reproductive cloning is quite low—only 3 to 5% of adult nuclei transferred to donor eggs result in live births. In addition, many clones that are born usually die soon thereafter of liver failure or infections. Many become oversized, a condition known as large offspring syndrome (LOS). In 2003, three of four cloned piglets developed to adulthood, but all three suddenly died of heart failure at less than six months of age. Dolly herself was euthanized at the relatively young age of six. Although she was put down because of virally induced lung The nucleus from a skin cell of a diabetic patient is removed.

Germ cells Some adult stem cells

Somatic cells

Figure 19.10 Methods to reprogram adult cell nuclei. 

Cells taken from adult organisms can be reprogrammed to pluripotent cells in a number of different ways. Nuclei from somatic cells can be transplanted into oocytes as during cloning. Somatic cells can be fused to ES cells created by some other means. Germ cells, and some adult stem cells, after prolonged culture appear to be reprogrammed. Recent work has shown that somatic cells in culture can be reprogrammed by introduction of defined factors.

cancer, she had been diagnosed with advanced-stage arthritis a year earlier. Thus, one difficulty in using genetic engineering and cloning to improve livestock is production of enough healthy animals.

Lack of imprinting One reason for these problems lies in a phenomenon discussed in chapter 13: genomic imprinting. Imprinted genes are expressed

The skin cell nucleus is inserted into the enucleated human egg cell.

Cell cleavage occurs as the embryo begins to develop in vitro.

The embryo reaches the blastocyst stage.

Inner cell mass

Diabetic patient

Early embryo

400 part III Genetic and Molecular Biology

ES cells

Blastocyst

differently depending on parental origin—that is, they are turned off in either egg or sperm, and this “setting” continues through development into the adult. Normal mammalian development depends on precise genomic imprinting. During normal development, there are a variety of epigenetic programming and reprogramming events that occur. The cloning protocol attempts to sidestep this and return the nucleus of a differentiated cell directly back to the state of a zygote. This involves changes in the state of the chromatin, and probably its organization as well. Until we understand all that this process involves, it is difficult to pinpoint the precise reasons for the low efficiency.

Nuclear reprogramming has been accomplished by use of defined factors Stimulated by the discovery of ES cells and success in the reproductive cloning of mammals, work turned to finding ways to reprogram adult cells to pluripotency without the use of embryos (figure 19.10). One approach was to fuse an ES cell to a differentiated cell. These fusion experiments showed that the nucleus of the differentiated cell could be reprogrammed by exposure to ES cell cytoplasm. Of course, the resulting cells are tetraploid (four copies of the genome), which limits their experimental and practical utility. Another line of research showed that primordial germ cells explanted into culture can give rise to cells that act similar to ES cells after extended time in culture. All of these different lines of inquiry showed that reprogramming of somatic nuclei was possible. Investigations into the characteristics of pluripotency identified a set of transcription factors that were active in ES cells. Then in 2006 Shinya Yamanaka and his coworkers showed that introducing genes that encode four of these transcription factors—Oct4, Sox2, c-Myc, and Klf4— could reprogram fibroblast cells in culture. Following introduction of the transcription factor genes, cells were selected that express a target gene regulated by Oct4 and Sox2, and these cells appeared to be pluripotent. These were named induced pluripotent stem cells, or iPS cells. The protocol has been refined by selection for another target gene known to be critical to the pluripotent state: Nanog. These Nanog-expressing iPS cells appear to be similar to ES cells in terms of developmental potential, as well as gene expression pattern. There is some indication that their chromatin structure, and thus their epigenetic state, may not be the same as that of ES cells.

It is worth asking what this work has taught us about the pluripotent state and the differentiated state. It is becoming clear that reprogramming is a multistep process. When this is done in culture, only a subset of cells make each step, thus explaining why the entire process is inefficient. Starting from a fibroblast, cells first change shape, becoming more spherical, and divide more rapidly. They then reverse part of their developmental program, becoming more like epithelial cells, a so-called mesenchyme-to-epithelial transition. Lastly, the stable expression of the core pluripotency regulatory factors Oct4, Sox2, and Nanog is established. The pluripotent state is maintained by a combination of transcription factors and chromatin structure (epigenetic changes). Whole-genome analysis of epigenetic markers has revealed changes in methylation and acetylation of histones, but the significance of specific modifications is not clear. In addition, iPS cells do not appear to have the same epigenetic signature as ES cells. The significance of this is also not clear. This technology has now been used to construct ES cells from patients with the inherited neurological disorder spinal muscular atrophy. These ES cells differentiate in culture into motor neurons that show the phenotype of the disease. The ability to derive ­disease-specific stem cells is an incredible advance for research on such diseases. This will allow us to study the cells affected by genetic diseases and to screen for possible therapeutics.

Pluripotent cells offer potential of tissue replacement Pluripotent cell types themselves have potential for therapeutic applications. One way to solve the problem of graft rejection, such as in skin grafts in severe burn cases, is to produce patient-specific lines of ES cells. The first method to accomplish this was called therapeutic cloning and it uses the same SCNT procedure that created Dolly to assemble an embryo. The nucleus is removed from a skin cell and inserted into an egg whose nucleus has already been removed. The egg with its skin cell nucleus is allowed to form a blastocyst-stage embryo. This artificial embryo is then used to derive ES cells for transfer to injured tissue (figure 19.11). Therapeutic cloning successfully addresses one key problem that must be solved before stem cells can be used to repair human tissues damaged by heart attack, nerve injury, diabetes, or Parkinson disease—the problem of immune acceptance. Since stem cells are cloned from a person’s own tissues, they pass the immune system’s “self” identity check, and the body readily accepts them. The

Figure 19.11 How human embryos might be used for therapeutic cloning.  In

Therapeutic Cloning Embryonic stem cells (ES cells) are extracted and grown in culture.

The stem cells are developed into healthy pancreatic islet cells needed by the patient.

Healthy pancreatic islet cells

The healthy tissue is injected or transplanted into the diabetic patient.

therapeutic cloning, after initial stages to reproductive cloning, the embryo is broken apart and its embryonic stem cells are extracted. These are grown in culture and used to replace the diseased tissue of the individual who provided the DNA. This is useful only if the disease in question is not genetic, as the stem cells are genetically identical to those of the patient.

Diabetic patient

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19 Cellular Mechanisms of Development 401

first clinical trial to use iPS cells, in which two patients were treated for age-related macular degeneration, was in Japan, and has run for over a year. The iPS cells were used to create a sheet of retinal pigmented epithelium that was transplanted into the patients. There was some small increase in light sensitivity, but more important, no serious adverse events were noted at 25 months. We are a long way from tissue engineering, but this is a first step in that direction.

Learning Outcomes Review 19.4

Cloning has long been practiced in plants. In animals, cells from early-stage embryos are also totipotent, but attempts to use adult nuclei for cloning led to mixed results. The nucleus of a differentiated cell requires reprogramming to be totipotent. This appears to be necessary at least in part because of genomic imprinting. Nuclei may be reprogrammed by fusion with an embryonic stem cell, which produces a tetraploid cell, or through the introduction of four important transcription factors. That reprogramming is possible was shown by reproductive cloning via somatic cell nuclear transfer (SCNT). In therapeutic cloning, the goal is to produce replacement tissue using a patient’s own cells. ■■

What changes must occur to produce a totipotent cell from a differentiated nucleus?

19.5

Pattern Formation

Learning Outcomes 1. Describe A/P axis formation in Drosophila. 2. Describe D/V axis formation in Drosophila.

For cells in multicellular organisms to differentiate into appropriate cell types, they must gain information about their relative locations in the body. All multicellular organisms seem to use positional information to determine the basic pattern of body compartments and, thus, the overall architecture of the adult body. This positional information then leads to intrinsic changes in gene activity, so that cells ultimately adopt a fate appropriate for their location. Pattern formation is an unfolding process. In the later stages, it may involve morphogenesis of organs, but during the earliest events of development, the basic body plan is laid down, along with the establishment of the anterior–posterior (A/P, head-to-tail) axis and the dorsal–­ventral (D/V, back-to-front) axis. Thus, pattern formation can be considered the process of taking a radially symmetrical cell and imposing two perpendicular axes to define the basic body plan, which in this way becomes bilaterally symmetrical. Developmental biologists use the term polarity to refer to the acquisition of axial differences in developing structures. The fruit fly Drosophila melanogaster is the best understood animal in terms of the genetic control of early patterning. We will concentrate on the Drosophila system here, and later, in chapter 52, we will examine axis formation in vertebrates in the context of their overall development. 402 part III Genetic and Molecular Biology

A hierarchy of gene expression that begins with maternally expressed genes controls the development of Drosophila. To understand the details of these gene interactions, we first need to briefly review the stages of Drosophila development.

Drosophila embryogenesis produces a segmented larva Drosophila and many other insects produce two different kinds of bodies during their development: the first, a tubular eating machine called a larva, and the second, an adult flying sex machine with legs and wings. The passage from one body form to the other, called metamorphosis, involves a radical shift in development (­figure 19.12). In this chapter, we concentrate on the process of going from a fertilized egg to a larva, which is termed embryogenesis.

Prefertilization maternal contribution The development of an insect like Drosophila begins before fertilization, with the construction of the egg. Specialized nurse cells that help the egg grow move some of their own maternally encoded mRNAs into the maturing oocyte (figure 19.12a). Following fertilization, the maternal mRNAs are transcribed into proteins, which initiate a cascade of sequential gene activations. Embryonic nuclei do not begin to function (that is, to direct new transcription of genes) until approximately 10 nuclear divisions have occurred. Therefore, the action of maternal, rather than zygotic, genes determines the initial course of Drosophila development.

Postfertilization events After fertilization, 12 rounds of nuclear division without cytokinesis produce about 4000 nuclei, all within a single cytoplasm. All of the nuclei within this syncytial blastoderm (figure 19.12b) can freely communicate with one another, but nuclei located in different sectors of the egg encounter different maternal products. Once the nuclei have spaced themselves evenly along the surface of the blastoderm, membranes grow between them to form the cellular blastoderm. Embryonic folding and primary­tissue development soon follow, in a process fundamentally similar to that seen in vertebrate development. Within a day of fertilization, embryogenesis creates a segmented, tubular body—which is destined to hatch out of the protective coats of the egg as a larva.

Morphogen gradients form the basic body axes in Drosophila Pattern formation in the early Drosophila embryo requires positional information encoded in labels that can be read by cells. The unraveling of this puzzle, work that earned the 1995 Nobel Prize for researchers Christiane Nüsslein-Volhard and Eric ­Wieschaus, is summarized in figure 19.13. We now know that two different genetic pathways control the establishment of A/P and D/V polarity in Drosophila.

Anterior–posterior axis Formation of the A/P axis begins during maturation of the oocyte and is based on opposing gradients of two different proteins: Bicoid and Nanos. These protein gradients are established by an interesting mechanism. Nurse cells in the ovary secrete maternally produced bicoid and nanos mRNAs into the maturing oocyte, where they are

Movement of maternal mRNA Follicle cells

Nurse cells Anterior

Oocyte

Posterior Nucleus

Fertilized egg

a.

Syncytial blastoderm

Cellular blastoderm

Nuclei line up along surface, and membranes grow between them to form a cellular blastoderm.

Segmented embryo prior to hatching

b. Hatching larva

Three larval stages

c.

differentially transported along microtubules to opposite poles of the oocyte (figure 19.14a). This differential transport comes about due to the use of different motor proteins to move the two mRNAs. The bicoid mRNA then becomes anchored in the cytoplasm at the end of the oocyte closest to the nurse cells, and this end will develop into the anterior end of the embryo. Nanos mRNA becomes anchored to the opposite end of the oocyte, which will become the posterior end of the embryo. Thus, by the end of oogenesis, the bicoid and nanos mRNAs are already set to function as cytoplasmic determinants in the fertilized egg (figure 19.14b). Following fertilization, translation of the anchored mRNA and diffusion of the proteins away from their respective sites of synthesis create opposing gradients of each protein: highest levels of Bicoid protein are at the anterior pole of the embryo (figure 19.15a), and highest levels of the Nanos protein are at the posterior pole. Concentration gradients of soluble molecules can specify different cell fates along an axis, and proteins that act in this way, like Bicoid and Nanos, are called m ­ orphogens. The importance of these morphogens can be seen by the effects of loss-of-function mutants: loss of Bicoid produces an embryo with only posterior sides, and loss of Nanos protein produces an embryo with only anterior sides. The Bicoid and Nanos proteins control the translation of two other maternal messages, hunchback and caudal, that encode transcription factors. Hunchback activates genes required for the formation of anterior structures, and Caudal activates genes required for the development of posterior (abdominal) structures. The hunchback and caudal mRNAs are evenly distributed across the egg (figure 19.15b), so how is it that proteins translated from these mRNAs become localized? The answer is that Bicoid protein binds to and inhibits translation of caudal mRNA. Therefore, caudal is translated only in the posterior regions of the egg where Bicoid is absent. Similarly, Nanos protein binds to and prevents translation of the hunchback mRNA. As a result, hunchback is translated only in the anterior regions of the egg (figure 19.15c). Thus, shortly after fertilization, four protein gradients exist in the embryo: anterior–posterior gradients of Bicoid and Hunchback proteins, and posterior–anterior gradients of Nanos and Caudal proteins (figure 19.15c).

Dorsal–ventral axis Metamorphosis

d. Thorax Head Abdomen

e.

Figure 19.12 The path of fruit fly development. Major

stages in the development of Drosophila melanogaster include formation of the (a) egg, (b) syncytial and cellular blastoderm, (c) larval instars, (d) pupa and metamorphosis into (e) a sexually mature adult.

The dorsal–ventral axis in Drosophila is established by actions of the dorsal gene product. Once again the process begins in the ovary, when maternal transcripts of the dorsal gene are put into the oocyte. However, unlike bicoid or nanos, the dorsal mRNA does not become asymmetrically localized. Instead, a series of steps are required for Dorsal to carry out its function. First, the oocyte nucleus, which is located to one side of the oocyte, synthesizes gurken mRNA. The gurken mRNA then accumulates in a crescent between the nucleus and the membrane on that side of the oocyte (figure 19.16a). This will be the future dorsal side of the embryo. The Gurken protein is a soluble cell-signaling molecule, and when it is translated and released from the oocyte, it binds to receptors in the membranes of the overlying follicle cells (figure 19.16b). These cells then differentiate into a dorsal morphology. Meanwhile, no Gurken signal is released from the other side of the oocyte, and the follicle cells on that side of the oocyte adopt a ventral fate. Following fertilization, a signaling molecule is differentially activated on the ventral surface of the embryo in a complex sequence chapter

19 Cellular Mechanisms of Development 403

Establishing the Polarity of the Embryo

Setting the Stage for Segmentation About 21/2 hours after fertilization, Bicoid protein turns on a series of brief signals from so-called gap genes. The gap proteins act to divide the embryo into large blocks. In this photo, fluorescent dyes in antibodies that bind to the gap proteins Krüppel (orange) and Hunchback (green) make the blocks visible; the region of overlap is yellow.

Fertilization of the egg triggers the production of Bicoid protein from maternal RNA in the egg. The Bicoid protein diffuses through the egg, forming a gradient. This gradient determines the polarity of the embryo, with the head and thorax developing in the zone of high concentration (green fluorescent dye in antibodies that bind Bicoid protein allows visualization of the gradient).

these fluorescent microscope images by 1995 Nobel laureate Christiane Nü ssleinVolhard and Sean Carroll, we watch a Drosophila egg pass through the early stages of development, in which the basic segmentation pattern of the embryo is established. The proteins in the photographs were made visible by binding fluorescent antibodies to each specific protein. (top)

©Steve Paddock and Sean Carroll; (bottom) ©Jim Langeland, Steve Paddock and Sean Carroll

500 μm

500 μm

Laying Down the Fundamental Regions About 0.5 hr later, the gap genes switch on the “pair-rule” genes, which are each expressed in seven stripes. This is shown for the pair-rule gene hairy. Some pair-rule genes are required only for even-numbered segments while others are required only for odd-numbered segments.

500 μm

Movement of maternal mRNA

Figure 19.13 Body organization in an early Drosophila embryo. In

Forming the Segments The final stage of segmentation occurs when a “segment polarity” gene called engrailed divides each of the seven regions into anterior and posterior compartments of the future segments. This occurs a little after the formation of the cellular blastoderm (see figure 19.12). The curved appearance of the embryo at this stage is because of a phenomenon called germ band extension that causes the embryo to fold over itself.

500 μm

Nucleus

bicoid mRNA moves toward anterior end Follicle cells

Nurse cells

Anterior

Anterior

Posterior

Posterior

Microtubules

a.

bicoid mRNA

nanos mRNA moves toward posterior end

nanos mRNA

b.

Figure 19.14  Specifying the A/P axis in Drosophila embryos I. a. In the ovary, nurse cells secrete maternal mRNAs into the

cytoplasm of the oocyte. Clusters of microtubules direct oocyte growth and maturation. Motor proteins travel along the microtubules transporting molecules in two directions. Bicoid mRNAs are transported toward the anterior pole of the oocyte, nanos mRNA is transported toward the posterior pole of the oocyte. b. A mature oocyte, showing localization of bicoid mRNAs to the anterior pole and nanos mRNAs to the posterior pole. 404 part III Genetic and Molecular Biology

Concentration

nanos mRNA hunchback mRNA bicoid mRNA caudal mRNA

Anterior

Posterior

a. Oocyte mRNAs

a.

400 μm

b.

400 μm

Anterior Bicoid protein

bicoid mRNA

caudal mRNA

Caudal protein

Posterior nanos mRNA

Nanos protein

hunchback mRNA

Hunchback protein

Concentration

b. After fertilization

Dorsal

Nanos protein Hunchback protein Bicoid protein Caudal protein

Wild-type embryo Anterior

Posterior

c. Early cleavage embryo proteins

Figure 19.15 Specifying the A/P axis in Drosophila embryos II. a. Unlike bicoid and nanos, hunchback and caudal

mRNAs are evenly distributed throughout the cytoplasm of the oocyte. b. Following fertilization, bicoid and nanos mRNAs are translated into protein, making opposing gradients of each protein. Bicoid binds to and represses translation of caudal mRNAs (in anterior regions of the egg). Nanos binds to and represses translation of hunchback mRNAs (in posterior regions of the egg). c. Translation of hunchback mRNAs in anterior regions of the egg will create a Hunchback gradient that mirrors the Bicoid gradient. Translation of caudal mRNAs in posterior regions of the embryo will create a Caudal gradient that mirrors the Nanos gradient.

of steps. This signaling molecule then binds to a membrane receptor in the ventral cells of the embryo and activates a signal transduction pathway in those cells. Activation of this pathway results in the selected transport of the Dorsal protein (which is everywhere) into

Ventral

c.

dorsal mutant 100 μm

Figure 19.16  Specifying the D/V axis in Drosophila embryos. a. The gurken mRNA (dark stain) is concentrated between the

oocyte nucleus (not visible) and the dorsal, anterior surface of the oocyte. b. In a more mature oocyte, Gurken protein (yellow stain) is secreted from the dorsal anterior surface of the oocyte, forming a gradient along the dorsal surface of the egg. Gurken then binds to membrane receptors in the overlying follicle cells. Double staining for actin (red) shows the cell boundaries of the oocyte, nurse cells, and follicle cells. c. For these images, cellular blastoderm–stage embryos were cut in cross section to visualize the nuclei of cells around the perimeter of the embryos. Dorsal protein (dark stain) is localized in nuclei on the ventral surface of the blastoderm in a wild-type embryo (left). The dorsal mutant on the right will not form ventral structures, and Dorsal is not present in ventral nuclei of this embryo. (a) Dr. Daniel St. Johnston; (b) ©Schupbach, T. and van Buskirk, C.; (c) ©From Roth et al., 1989, courtesy of Siegfried Roth

ventral nuclei, forming a gradient along the D/V axis. The Dorsal protein levels are highest in the nuclei of ventral cells (figure 19.16c). (Note that many Drosophila genes are named for the mutant phenotype that results from a loss of function in that gene. A lack chapter

19 Cellular Mechanisms of Development 405

Figure 19.17 Mutations in homeotic genes. The combination of mutations in three different homeotic genes causes a complete conversion of the third thoracic segment to the second. As only the second thoracic segment has wings, this results in a four-winged fly.

David Scharf/Science Source

of dorsal function produces dorsalized embryos with no ventral structures.) The Dorsal protein is a transcription factor, and once it is transported into nuclei, it activates genes required for the proper development of ventral structures, simultaneously repressing genes that specify dorsal structures. Hence, the product of the dorsal gene ultimately directs the development of ventral structures. Although profoundly different mechanisms are involved, the unifying factor controlling the establishment of both A/P and D/V polarity in Drosophila is that bicoid, nanos, gurken, and dorsal are all maternally expressed genes. The polarity of the future embryo in both instances is therefore laid down in the oocyte using information coming from the maternal genome. The preceding discussion simplifies events, but the outline is clear: polarity is established by the creation of morphogen gradients in the embryo based on maternal information in the egg. These gradients then drive the expression of the zygotic genes that will actually pattern the embryo. This reliance on a hierarchy of regulatory genes is a unifying theme for all of development.

The body plan is produced by sequential activation of genes Let us now return to the process of pattern formation in Drosophila along the A/P axis. Determination of structures is accomplished by the sequential activation of three classes of segmentation genes. These genes create the hallmark segmented body plan of a fly, which consists of three fused head segments, three thoracic segments, and eight abdominal segments (see figure 19.12e). To begin, Bicoid protein exerts its profound effect on the organization of the embryo by activating the translation and transcription of hunchback mRNA (which is the first mRNA to be transcribed after fertilization). Hunchback is a member of a group of nine genes called the gap genes. These genes map out the initial subdivision of the embryo along the A/P axis (see figure 19.13). 406 part III Genetic and Molecular Biology

All of the gap genes encode transcription factors, which, in turn, regulate the expression of eight or more pair-rule genes. Each of the pair-rule genes, such as hairy, produces seven distinct bands of protein, which appear as stripes when visualized with fluorescent reagents (see figure 19.13). These bands subdivide the broad gap regions and establish boundaries that divide the embryo into seven zones. When mutated, each of the pair-rule genes alters every other body segment. All of the pair-rule genes also encode transcription factors, and they, in turn, regulate the expression of each other and of a group of nine or more segment polarity genes. The segment polarity genes are each expressed in 14 distinct bands of cells, which subdivide each of the seven zones specified by the pair-rule genes (see figure 19.13). The engrailed gene, for example, divides each of the seven zones established by hairy into anterior and posterior compartments. The segment polarity genes encode proteins that function in cell–cell signaling pathways. Thus, they function in inductive events—which occur after the syncytial blastoderm is divided into cells—to fix the anterior and posterior fates of cells within each segment. In summary, within 3 hr after fertilization, a highly orchestrated cascade of segmentation gene activity transforms the broad gradients of the early embryo into a periodic, segmented structure with A/P and D/V polarity. The activation of the segmentation genes depends on the free diffusion of maternally encoded morphogens, which is possible only within the syncytial blastoderm of the early ­Drosophila embryo.

Segment identity arises from the action of homeotic genes The cascade of gene expression just described produces a segmented larva. The larval segments appear similar, but they can be distinguished as head, thoracic, and abdominal segments. In the adult, the segments are much more clearly defined. The identity of these segments depends on the action of a set of genes called homeotic genes. This curious name has to do with the phenotype produced by mutants in these genes: they cause transformation of one body part to another, called homeosis. Perhaps the most striking of these is found in the mutant Antennapedia, which causes a transformation of antenna to legs, resulting in a fly with legs on its head! The first homeotic gene identified was Ultrabithorax, which causes a transformation of part of the third thoracic segment (T3) to a second thoracic segment (T2). This gene is actually part of a group of homeotic genes affecting the thoracic and abdominal segments. A fly carrying mutations in three of these genes will actually have a complete T3-to-T2 conversion. Because T2 has wings, and T3 does not, the result is a 4-winged fly (figure 19.17). The common ancestor to all insects had four wings, so the homeotic genes are actually preventing the formation of the second set of wings. The expression of these homeotic genes is controlled by the cascade of gene expression that produced the segmented larvae.

The homeotic genes in turn regulate the expression of genes needed to produce segment identity. We will consider these genes in more detail in the next section, examining the evolution of pattern formation, as they prove to be of universal importance.

Learning Outcomes Review 19.5

Pattern formation in animals involves the coordinated expression of a hierarchy of genes. Gradients of morphogens in Drosophila specify A/P and D/V axes, then lead to sequential activation of segmentation genes. Bicoid and Nanos protein gradients determine the A/P axis. The protein Dorsal determines the D/V axis, but activation requires a series of steps beginning with the oocyte’s Gurken protein. The action of homeotic genes provides segment identity. Does the individual carrying a maternal-effect mutation show any phenotype?

19.6 Evolution

of Pattern Formation

Learning Outcomes 1. Identify genes likely to affect morphology. 2. Explain the importance of Hox genes in evolution. 3. Discuss mechanisms that can change development.

This can be seen even in simple organisms like sea urchins, part of a diverse group of echinoderms called Echinoidea (see chapter 34), which includes sea urchins and sand dollars. Species of Echinoidea exhibit two different forms of development: one includes an intermediate larval stage, and the other omits this larval stage (figure 19.18). Indirectly developing species go through a larval stage, called a pluteus, which undergoes metamorphosis to produce the adult form. In contrast, direct developers skip the pluteus stage and embryogenesis produces a smaller adult form. Phylogenetic analysis (see chapter 23) indicates that indirect development is the primitive state. One possible explanation for these different patterns of development is that the direct developers simply lack the necessary genes to make the larval form. This turns out not to be the case, as comparisons of the genomes of direct and indirect developers reveal they have essentially the same set of genes. So, the differences seen in development are due to differences in the pattern of gene regulatory networks.

Direct Development

Egg and sperm released

Eggs brooded by female

MEIOSIS

Indirect Development

R FE n Adult (benthic)

Changes in gene expression produce phenotypic changes

TI

LI

TI ZA

MEIOSIS

■■

Most species exhibit significant phenotypic diversity. This diversity can arise from changes to the protein-coding regions of many genes. A much smaller amount of DNA sequence is devoted to controlling gene expression, but mutations to these sequences, and the proteins that interact with them, can have a large effect on phenotype. This is particularly important when changes in gene expression affect development. Differences between related species are often due to genetic changes that produce differences in how the species develop.

ON

R FE

Development of free-swimming larva

n Adult (benthic)

2n

2n

TI

LI

TI ZA

ON

Direct development into adult

Pluteus larva (plankton) Juvenile

Metamorphosis and settlement to seafloor

Juvenile

Figure 19.18  Direct and indirect sea urchin development.  Indirect development goes through a larval stage that direct development omits.

chapter

19 Cellular Mechanisms of Development 407

Drosophila HOM Chromosomes

Mouse Hox Chromosomes

Drosophila HOM genes

Hox 1

Antennapedia complex

Bithorax complex

Hox 2

lab

Ubx abd-A abd-B

Hox 3

pb

Dfd Scr Antp

Head

Thorax

Hox 4

Abdomen

Fruit fly embryo

Mouse embryo

Fruit fly

Mouse

a.

b.

Figure 19.19 A comparison of homeotic gene clusters in the fruit fly Drosophila melanogaster and the mouse Mus musculus. a. Drosophila homeotic genes. Called the homeotic gene complex, or HOM complex, the genes are grouped into two clusters: the

Antennapedia complex (anterior) and the bithorax complex (posterior). b. The Drosophila HOM genes and the mouse Hox genes are related genes that control the regional differentiation of body parts in both animals. These genes are located on a single chromosome in the fly and on four separate chromosomes in mammals. In this illustration, the genes are color-coded to match the parts of the body along the A/P axis in which they are expressed. Note that the order of the genes along the chromosome(s) is mirrored by their pattern of expression in the embryo and in structures in the adult fly.

Pattern formation is controlled by a small number of ancient genes We saw in the last section that the basic body plan in Drosophila is laid down by a cascade of gene expression to specify A/P and D/V axes. Further, the adult form is affected by genes that specify the structures formed in different segments. The action of these homeotic genes is indicated by mutations that produce normal body parts in inappropriate places. While this is of inherent interest, the real surprise is that related genes are found in essentially all animals.

Homeotic gene complexes In the early 1950s, geneticist and Nobel laureate Edward Lewis discovered that several homeotic genes, including Ultrabithorax, map together on the third chromosome of Drosophila in a tight cluster called the bithorax complex. Mutations in these genes all affect body parts of the thoracic and abdominal segments, and Lewis concluded that the genes of the bithorax complex control the development of body parts in the rear half of the thorax and all of the abdomen. Interestingly, the order of the genes in the bithorax complex mirrors the order of the body parts they control, as though the genes are activated serially. Genes at the beginning of the cluster switch on development of the thorax; those in the middle control the anterior part of the abdomen; and those at the end affect the posterior tip of the abdomen. 408 part III Genetic and Molecular Biology

A second cluster of homeotic genes, the Antennapedia complex, was discovered in 1980 by Thomas Kaufman. The Antennapedia complex governs the anterior end of the fly, and the order of genes in this complex also corresponds to the order of segments they control (figure 19.19a).

The homeobox When the genes in the bithorax and Antennapedia complexes were cloned, a very interesting observation was made. These genes all contain a conserved sequence of 180 nucleotides that codes for a 60–amino acid, DNA-binding domain. As this domain was found in homeotic genes, it was named the homeodomain, and the DNA that encodes it is called the homeobox. Homeobox-containing genes were found in virtually every animal examined, and they were found in clusters as well. This led to the term Hox genes, which refers to a homeobox-containing gene that specifies the identity of a body part. These genes function as transcription factors, with DNA-binding domains encoded by a homeobox. The discovery of Hox gene clusters in animals was an important unifying observation about animal development. Hox clusters are regions of the genome that are important for pattern formation, and the genes that are the targets of these transcription factors affect the cell behaviors associated with organ morphogenesis.

Evolution of homeobox-containing genes A large amount of research has led to a unified view of homeotic gene evolution. It is now clear that the Drosophila bithorax and Antennapedia complexes are actually two parts of a single group of genes. The intact Hox gene cluster, which is observed in most lineages, probably arose through the tandem duplication of an ancient homeobox-containing gene. The number of clusters varies in different lineages; for example, in vertebrates there are four Hox gene clusters. As in Drosophila, the spatial domains of Hox gene expression correlate with the order of the genes on the chromosome (figure 19.19b). The existence of four Hox clusters in vertebrates is thought to indicate that there have been two whole-genome duplications in the vertebrate lineage. This raises the issue of when the original cluster arose. To address this question, researchers have turned to a more primitive chordate, Amphioxus (now called Branchiostoma; see chapter 34). A single cluster of Hox genes was found in Amphioxus, which implies that indeed there have been two duplications in the vertebrate lineage, at least of the Hox cluster. Given the single cluster in arthropods, this finding implies that the common ancestor to all animals with bilateral symmetry had a single Hox cluster as well. This has been extended to even more primitive animals: the radially symmetrical cnidarians such as Hydra (see chapter 33). Thus far, Hox genes have been found in a number of cnidarian species, and these may be arranged into clusters, but more related species need to be examined. However, it appears likely that the appearance of the ancestral Hox cluster likely preceded the evolution of bilateral symmetry in animal evolution.

Pattern formation in plants is also under genetic control The evolutionary split between plant and animal cell lineages occurred about 1.6 bya, before the appearance of multicellular organisms with defined body plans. This implies that multicellularity evolved independently in plants and animals. Because of the activity of meristems, additional modules can be added to plant bodies throughout their lifetimes. In addition, plant flowers and roots have a radial organization, in contrast to the bilateral symmetry of most animals. We may therefore expect that the genetic control of pattern formation in plants is fundamentally different from that of animals. Although plants have homeobox-containing genes, they are not organized into complexes of Hox genes similar to the ones that determine regional identity of developing structures in animals. Instead, the predominant homeotic gene family in plants appears to be the MADS box genes. MADS box genes are a family of transcriptional regulators found in most eukaryotic organisms, including plants, animals, and fungi. The MADS box is a conserved DNA-binding and dimerization domain, named after the first five genes to be discovered with this domain. Only a small number of MADS box genes are found in animals, where their functions include the control of cell proliferation and tissue-specific gene expression in postmitotic muscle cells. They do not appear to play a role in the patterning of animal embryos. In contrast, the number and functional diversity of MADS box genes increased considerably during the evolution of land plants, and there are more than 100 MADS box genes in the Arabidopsis genome. In flowering plants, the MADS box genes dominate the control of

development, regulating such processes as the transition from vegetative to reproductive growth, root development, and floral organ identity. Although distinct from genes in the Hox clusters of animals, plant Hox genes encode transcription factors that have important developmental functions. One such example is the family of knottedlike homeobox (knox) genes, which are important regulators of shoot apical meristem development in both seed-bearing and non-seed-bearing plants. Mutations that affect expression of knox genes produce changes in leaf and petal shape, suggesting that these genes play an important role in generating leaf form.

Developmental mechanisms evolve Understanding how development evolves requires integration of knowledge about genes, gene expression, development, and evolution. Either transcription factors or signaling molecules can be modified during evolution, changing the timing or position of gene expression and, as a result, gene function.

Heterochrony Genetic changes that cause alterations in developmental timing are called heterochrony. A heterochronic mutation might affect a gene that controls the transition from juvenile to adult stage, when it can produce reproductive organs. A mutation in a gene that controls flowering time in plants can result in a small plant that flowers quickly rather than requiring months or years of growth before it flowers. Most mutations that affect developmental regulatory genes are lethal, but rarely a novel phenotype emerges with increased fitness that persists. If a mutation leading to early flowering increases the fitness of a plant, its allele frequency should increase (see chapter 20). For example, a tundra plant that flowers earlier, enabling it to be fertilized and set seed quickly, could have increased fitness over an individual of the same species that flowers later, just as the short summer comes to a close.

Homeosis As discussed earlier, changes in the spatial pattern of gene expression can result in homeosis, which occurs when one body segment takes on the identity of another. The four-winged Drosophila fly in figure 19.17 is an example of a change in gene expression pattern with a dramatic effect on morphology. This phenotype resembles more ancestral insects with four wings rather than two. The Antennapedia mutant, which has a leg on its head where an antenna should be, is a dominant mutation that is immediately visible. Mutations of this sort can arise spontaneously in the natural world or by mutagenesis in the laboratory. Although neither of these examples produces a phenotype that is advantageous, it is possible such a mutation could produce a new phenotype that provides a fitness advantage, and thus would evolve by natural selection.

Changes in transcription The timing or location of gene expression can be modified in several ways, giving rise to heterochrony or homeosis. The coding sequence of a gene can contain multiple regions with different functions. A mutation in a DNA-binding motif, such as the MADS box domain or homeodomain, can alter how a protein binds to target genes. This could alter a developmental pathway, or result in the complete loss of the pathway. Alternatively, the modified chapter

19 Cellular Mechanisms of Development 409

transcription factor might bind to a different target and initiate a new sequence of developmental events. Mutations in the regulatory regions of genes would also affect patterns of transcription. If the regulatory region of a gene encoding a transcription factor were altered, this could change an entire developmental pathway. Changes to the promoter or enhancer of a transcription factor gene could prevent expression of the gene, or result in different timing or tissue-specific expression without changing the targets of the transcription factor.

Changes in signaling pathways Signaling pathways help cells coordinate information about neighboring cells and the external environment that is essential for successful development. If the structure of a ligand changes, it may no longer bind to its target receptor; it could bind to a different receptor or no receptor at all. If, as a result of a genetic change, a receptor is produced in a different cell type, a homeotic phenotype may appear. A small change in signaling molecules can alter their targets.

Learning Outcomes Review 19.6

Direct development arose by changes in gene expression. Hox genes are characterized by a DNA-binding domain encoded by a homeodomain. These genes affect development in essentially all animals. There are four clusters in vertebrates from the duplication of a single ancestral cluster. Plants have Hox genes, but use MADS box genes for pattern formation. Changes in levels, patterns, or timing of gene expression can affect morphology. ■■

What would it say about the evolution of pattern formation if plants had Hox clusters similar to those in animals?

19.7

Morphogenesis

Learning Outcomes 1. Discuss the importance of cell shape changes and cell migration in development. 2. Explain how cell death can contribute to morphogenesis. 3. Describe the role of the extracellular matrix in cell migration.

At the end of cleavage, the Drosophila embryo still has a relatively simple structure: it comprises several thousand ­identical-looking cells, which are present in a single layer surrounding a central yolky region. The next step in embryonic development is morphogenesis—the generation of ordered form and structure. Morphogenesis is the product of changes in cell structure and cell behavior. Animals regulate the following processes to achieve morphogenesis: ■■ ■■ ■■ ■■ ■■

the number, timing, and orientation of cell divisions; cell growth and expansion; changes in cell shape; cell migration; and cell death.

410 part III Genetic and Molecular Biology

Plant and animal cells are fundamentally different in that animal cells have flexible surfaces and can move, but plant cells are immotile and encased within stiff cellulose walls. Each cell in a plant is fixed into position when it is created. Thus, animal cells use cell migration extensively during development, whereas plants use the other four mechanisms but lack cell migration. We consider the morphogenetic changes in animals here, and plant morphogenesis is detailed in chapter 40.

Cell division during development may result in unequal cytokinesis The orientation of the mitotic spindle determines the plane of cell division in eukaryotic cells. The coordinated function of microtubules and their motor proteins determines the respective position of the mitotic spindle within a cell (see ­chapter 10). If the spindle is centrally located in the dividing cell, two equal-sized daughter cells will result. If the spindle is off to one side, one large daughter cell and one small daughter cell will result. The great diversity of cleavage patterns in animal embryos­is determined by differences in spindle placement. In many cases, the fate of a cell is determined by its relative placement in the embryo during cleavage. For example, in preimplantation mammalian embryos, cells on the outside of the embryo usually differentiate into trophectoderm cells, which form only extraembryonic structures later in development (for example, a part of the placenta). In contrast, the embryo proper is derived from the inner cell mass, cells which, as the name implies, are in the interior of the embryo.

Cells change shape and size as morphogenesis proceeds In animals, cell differentiation is often accompanied by profound changes in cell size and shape. For example, the large nerve cells that connect your spinal cord to the muscles in your big toe develop long processes called axons that span this entire distance. The cytoplasm of an axon contains microtubules, which are used for motor-driven transport of materials along the length of the axon. As another example, muscle cells begin as myoblasts, undifferentiated muscle precursor cells. They eventually undergo conversion into the large, multinucleated muscle fibers that make up mammalian skeletal muscles. These changes begin with the expression of the MyoD1 gene, which encodes a transcription factor that binds to the promoters of muscle-determining genes to initiate these changes.

Programmed cell death is a necessary part of development Not every cell produced during development is destined to survive. For example, human embryos have webbed fingers and toes at an early stage of development. The cells that make up the webbing die in the normal course of morphogenesis. As another example, vertebrate embryos produce a very large number of neurons, ensuring that enough neurons are available to make the necessary synaptic connections, but over half of these neurons never make connections and die in an orderly way as the nervous system develops. Unlike accidental cell deaths due to injury, these cell deaths are planned—and indeed required—for proper development and

Organism

Caenorhabditis elegans

Inhibitor:

CED-9

Activator:

CED-4

Apaf1

Apoptotic Protease:

CED-3

Caspase-8 or -9

Apoptosis

Apoptosis

Inhibition Activation

a.

Mammalian Cell

Inhibitor

Bcl-2

b.

Figure 19.20 Programmed cell death pathway. Apoptosis, or programmed cell death, is necessary for the normal development of all animals. a. In the developing nematode, for example, two genes, ced-3 and ced-4, code for proteins that cause the programmed cell death of 131 specific cells. In the other (surviving) cells of the developing nematode, the product of a third gene, ced-9, represses the death program encoded by ced-3 and ced-4. b. The mammalian homologues of the apoptotic genes in C. elegans are bcl-2 (ced-9 homologue), Apaf1 (ced-4 homologue), and caspase-8 or -9 (ced-3 homologues). In the absence of any cell survival factor, Bcl-2 is inhibited and apoptosis occurs. In the presence of nerve growth factor (NGF) and NGF receptor binding, Bcl-2 is activated, thereby inhibiting apoptosis. morphogenesis. Cells that die due to injury typically swell and burst, releasing their contents into the extracellular fluid. This form of cell death is called necrosis. In contrast, cells programmed to die shrivel and shrink in a process called apoptosis, which means “falling away,” and their remains are taken up by surrounding cells.

Genetic control of apoptosis Apoptosis occurs when a “death program” is activated. All animal cells appear to possess such programs. In C. elegans, the same 131 cells always die during development in a predictable and reproducible pattern. Work on C. elegans showed that three genes are central to this process. Two (ced-3 and ced-4) activate the death program itself; if either is mutant, those 131 cells do not die, and go on instead to form nervous tissue and other tissue. The third gene (ced-9) represses the death program encoded by the other two: all 1090 cells of the C. elegans embryo die in ced-9 mutants. In ced-9/ced-3 double mutants, all 1090 cells live, which suggests that ced-9 inhibits cell death by functioning prior to ced-3 in the apoptotic pathway (figure 19.20a). The mechanism of apoptosis appears to have been highly conserved during the course of animal evolution. In human nerve cells, the Apaf1 gene is similar to ced-4 of C. elegans and activates the cell death program, and the human bcl-2 gene acts similarly to ced-9 to repress apoptosis. If a copy of the human bcl-2 gene is transferred into a nematode with a defective ced-9 gene, bcl-2 suppresses the cell death program of ced-3 and ­ced-4.

The mechanism of apoptosis The product of the C. elegans ced-4 gene is a protease that activates the product of the ced-3 gene, which is also a protease. The human

Apaf1 gene is actually named for its role: Apoptotic protease activating factor. It activates two proteases called caspases that have a role similar to the Ced-3 protease in C. elegans (­figure  19.20b). When the final proteases are activated, they chew up proteins in important cellular structures such as the cytoskeleton and the nuclear lamina, leading to cell fragmentation. The role of Ced-9/Bcl-2 is to inhibit this program. Specifically, it inhibits the activating protease, preventing the activation of the destructive proteases. The entire process is thus controlled by an inhibitor of the death program. Both internal and external signals control the state of the Ced-9/Bcl-2 inhibitor. For example, in the human nervous system, neurons have a cytoplasmic inhibitor of Bcl-2 that allows the death program to proceed (see figure 19.20b). In the presence of nerve growth factor, a signal transduction pathway leads to the cytoplasmic inhibitor being inactivated, allowing Bcl-2 to inhibit apoptosis and the nerve cell to survive.

Cell migration gets the right cells to the right places The migration of cells is important during many stages of animal development. The movement of cells involves both adhesion and the loss of adhesion. Adhesion is necessary for cells to get “traction,” but cells that are initially attached to others must lose this adhesion to be able to leave a site. Cell movement also involves cell-to-substrate interactions, and the extracellular matrix may control the extent or route of cell migration. The central paradigm of morphogenetic­cell movements in animals is a change in cell adhesiveness, which is mediated by changes in the composition of macromolecules in the plasma membranes of cells chapter

19 Cellular Mechanisms of Development 411

or in the extracellular matrix. Cell-to-cell interactions are often mediated through cadherins, but cell-to-substrate interactions often involve integrin-to-­extracellular-matrix (ECM) interactions.

SCIENTIFIC THINKING

Cadherins

gastrulation should prevent cell movement.

Cadherins are a large gene family, with over 80 members identified in humans. In the genomes of Drosophila, C. elegans, and humans, the cadherins can be sorted into several subfamilies that exist in all three genomes. The cadherin proteins are all transmembrane proteins that share a common motif, the cadherin domain, a 110-­amino-acid domain in the extracellular portion of the protein that ­mediates Ca2+dependent binding between like cadherins (­homophilic binding). Experiments in which cells are allowed to sort in vitro illustrate the function of cadherins. Cells with the same cadherins adhere specifically to one another, while not adhering to other cells with different cadherins. If cell populations with different cadherins are dispersed and then allowed to reaggregate, they sort into two populations of cells based on the nature of the cadherins on their surface. An example of the action of cadherins can be seen in the development of the vertebrate nervous system. All surface ectoderm cells of the embryo express E-cadherin. The formation of the nervous system begins when a central strip of cells on the dorsal surface of the embryo turns off E-cadherin expression and turns on N-cadherin expression. In the process of neurulation, the formation of the neural tube (see chapter  52), the central strip of N-cadherin-expressing cells folds up to form the tube. The neural tube pinches off from the overlying cells, which continue to express E-cadherin. The surface cells outside the tube differentiate into the epidermis of the skin, whereas the neural tube develops into the brain and spinal cord of the embryo.

Integrins In some tissues, such as connective tissue, much of the volume of the tissue is taken up by the spaces between cells. These spaces are filled with a network of molecules secreted by surrounding cells, termed a matrix. In connective tissue such as cartilage, long polysaccharide chains are covalently linked to proteins (proteoglycans), within which are embedded strands of fibrous protein (collagen, elastin, and fibronectin). Migrating cells traverse this matrix by binding to it with cell surface proteins called integrins. Integrins are attached to actin filaments of the cytoskeleton and protrude out from the cell surface in pairs, like two hands. The “hands” grasp a specific component of the matrix, such as collagen or fibronectin, thus linking the cytoskeleton to the fibers of the matrix. In addition to providing an anchor, this binding can initiate changes within the cell, alter the growth of the cytoskeleton, and activate gene expression and the production of new proteins. The process of gastrulation (described in detail in ­chapter 52), during which the hollow ball of animal embryonic cells folds in on itself to form a multilayered structure, depends on fibronectin–integrin interactions. For example, injection of antibodies against either fibronectin or integrins into salamander embryos blocks binding of cells to fibronectin in the ECM and inhibits gastrulation. The result is like a huge traffic jam following a major accident on a freeway: cells (cars) keep com412 part III Genetic and Molecular Biology

Hypothesis: Fibronectin is required for cell migration during gastrulation. Prediction: Blocking fibronectin with antifibronectin antibodies before Test: Staged salamander embryos were injected either with antifibronectin antibody, or with preimmune serum as a control, prior to gastrulation. Cell movements were then monitored photographically. Treated with Preimmune

Treated with Antifibronectin

Blastopore

Cells have moved into the interior

Blastopore

285 μm

a.

Cells pile up on the surface

285 μm

b.

Result: The experimental embryos injected with antifibronectin antibody show extremely aberrant gastrulation where cells pile up and do not enter the interior of the embryo. Control embryos gastrulate normally. Conclusion: Fibronectin is required for cells to migrate into the interior of the embryo during gastrulation. Further Experiments: How can this same system be used to analyze the role of fibronectin in other early morphogenetic events?

Figure 19.21  Fibronectin is necessary for cell migration during gastrulation. From Boucaut et al., 1984, courtesy of J-C Boucaut ing, but they get backed up since they cannot get beyond the area of inhibition (accident site) (figure 19.21). Similarly, a targeted knockout of the fibronectin gene in mice resulted in gross defects in the migration, proliferation, and differentiation of embryonic mesoderm cells. Thus, cell migration is largely a matter of changing patterns of cell adhesion. As a migrating cell travels, it continually extends projections that probe the nature of its environment. Tugged this way and that by different tentative attachments, the cell literally feels its way toward its ultimate target site.

Learning Outcomes Review 19.7

Morphogenesis is the generation of ordered form and structure. This process proceeds along with cell differentiation. The primary mechanisms of morphogenesis are cell shape change and cell migration. Apoptosis is programmed cell death that is a necessary part of morphogenesis. Cell migration in animals involves alternating changes in adhesion brought about by cadherins and integrins. ■■

Why is cell death important to morphogenesis?

Chapter Review Science Photo Library/Steve Gschmeissner/Getty Images

19.1 The Process of Development Development is the sequence of systematic, gene-directed changes throughout a life cycle. The four subprocesses of development are cell division, cell differentiation, pattern formation, and morphogenesis.

19.2 Cell Division Development begins with cell division. In animals, cleavage stage divisions divide the fertilized egg into numerous smaller cells called blastomeres. During cleavage the G1 and G2 phases of the cell cycle are shortened or eliminated (figure 19.2). Every cell division is known in the development of C. elegans. The lineage of 959 adult somatic Caenorhabditis elegans cells is invariant. Knowledge of the differentiation sequence and outcome allows study of developmental mechanisms. Plant growth occurs in specific areas called meristems. Plant growth continues throughout the life span from meristematic stem cells that can divide and differentiate into any plant tissue.

19.3 Cell Differentiation Cells become determined prior to differentiation. The process of determination commits a cell to a particular developmental pathway prior to its differentiation. This is not visible but can be tracked experimentally. Determination is due to differential inheritance of cytoplasmic factors or cell-to-cell interactions. Determination can be due to cytoplasmic determinants. In tunicates, determination of tail muscle cells depends on the presence of mRNA for the Macho-1 transcription factor, which is deposited in the egg cytoplasm during gamete formation. Induction can lead to cell differentiation. Induction occurs when one cell type produces signal molecules that induce gene expression in neighboring target cells. In frogs, cells from animal and vegetal poles do not develop into mesoderm when isolated. In tunicates, signaling by the growth factor FGF induces mesoderm development. Stem cells can divide and produce cells that differentiate. Stem cells replace themselves by division and produce cells that differentiate. Totipotent stem cells can give rise to any cell type, including extraembryonic tissues; pluripotent cells can give rise to all cells of an organism; and multipotent stem cells can give rise to many kinds of cells. Embryonic stem cells are pluripotent cells derived from embryos. Embryonic stem cells are derived from the inner cell mass of the blastocyst (figure 19.8). They can differentiate into any adult tissue in a mouse.

19.4 Nuclear Reprogramming Reversal of determination has allowed cloning. Cells undergo no irreversible changes during development. However, transplanted nuclei from older donors are less able to direct

complete development. The cloning of the sheep Dolly showed that the nucleus of an adult cell can be reprogrammed to be totipotent (figure 19.9). Reproductive cloning has inherent problems. Reproductive cloning has a low success rate, and clones often develop age-associated diseases. Cloning is inefficient because of the difficulty of reprogramming epigenetic modifications. Nuclear reprogramming has been accomplished by use of defined factors. Adult cells can be converted into pluripotent cells by introduction of four genes for transcription factors. These induced pluripotent cells appear to be similar to ES cells. Pluripotent cells offer potential of tissue replacement The use of cells cloned from a patient’s cells to replace damaged tissue could avoid the problem of transplant rejection. Clinical trials are underway to treat a form of blindness using iPS cells.

19.5 Pattern Formation Drosophila embryogenesis produces a segmented larva. The maternal contribution of mRNA along with the postfertilization events of cellular blastoderm formation produce a segmented embryo. Morphogen gradients form the basic body axes in Drosophila. Pattern formation produces two perpendicular axes in a bilaterally symmetrical organism. Positional information leads to changes in gene activity so cells adopt a fate appropriate for their location. Formation of the anterior–posterior (A/P) axis is based on opposing gradients of morphogens, Bicoid and Nanos, synthesized from maternal mRNA (figures 19.14, & 19.15). The dorsal–ventral (D/V) axis is established by a gradient of the Dorsal transcription factor. Successive action of transcription factors divides the embryo into segments. The body plan is produced by sequential activation of genes. Segment identity arises from the action of homeotic genes. Mutations in homeotic genes cause transformations of one body part for another. These genes control segment identity.

19.6 Evolution of Pattern Formation Changes in gene expression produce phenotypic changes. Direct versus indirect development in sea urchins is due to changes in gene expression and not to different sets of genes in related species. Pattern formation is controlled by a small number of ancient genes. Hox genes in Drosophila are found in two clusters: the Antennapedia complex and the bithorax complex. These genes have a homeobox that encodes a DNA-binding domain. In vertebrates, related genes are found in a single complex called a Hox gene cluster. There are 4 such clusters in vertebrates. Pattern formation in plants is also under genetic control. Plants have both Hox genes and MADS box genes. Hox genes have developmental functions but MADS box genes act in patterning more like Hox genes in animals.

chapter

19 Cellular Mechanisms of Development 413

Developmental mechanisms evolve. Heterochrony is a change in developmental timing. Homeosis changes patterns of gene expression to alter developmental fate. Changes in patterns of transcription or signaling pathways can alter development.

Programmed cell death is a necessary part of development. Apoptosis, the programmed death of cells, removes structures once they are no longer needed (figure 19.20). Cell migration gets the right cells to the right places. The migration of cells requires both adhesion and loss of adhesion between cells and their substrate. Cell-to-cell interactions are often mediated by cadherin proteins, whereas cell-to-substrate interactions may involve integrin-to-extracellular-matrix interactions. Integrins bind to fibers found in the extracellular matrix. This action can alter the cytoskeleton and activate gene expression.

19.7 Morphogenesis Cell division during development may result in unequal cytokinesis. Cells change shape and size as morphogenesis proceeds. Depending on the orientation of the mitotic spindle, cells of equal or different sizes can arise. Morphogenesis involves changes in cell shape and size and cell migration.

Visual Summary Development Cell division

preceded by

in Animals cleavage to

Blastomeres

due to

undergo

produce

Induction

reverse by

Nuclear reprogramming

Evolution

requires into

Morphogen gradients

Epigenetic changes

Cell–cell interactions

formation of Totipotent Pluripotent Multipotent Unipotent Meristems

involves

Stem cells

Cytoplasmic determinants

Morphogenesis

involves

from

Determination

example Plants

Pattern formation

Differentiation

duplication of Hox genes

Anterior– posterior axis

Dorsal– ventral axis

Phenotypic changes

414 part III Genetic and Molecular Biology

Regulation

Cell division Cell shape Cell migration Apoptosis Cell death Necrosis

due to Segmentation genes

Extracellular matrix

example Embryonic

of

Segment identity

due to

Homeotic genes

Cadherins Integrins

Review Questions Science Photo Library/Steve Gschmeissner/Getty Images

U N D E R S TA N D 1. During development, cells become a. b. c. d.

differentiated before they become determined. determined before they become differentiated. determined by the loss of genetic material. differentiated by the loss of genetic material.

2. Determination can occur by a. b. c. d.

the action of cytoplasmic determinants. induction by other cells. the loss of chromosomes during cell division. Both a and b are correct.

3. The rapid divisions that occur early in development are made possible by shortening a. M phase. b. S phase.

c. G1 and G2 phases. d. All of the choices are correct.

4. A pluripotent cell is one that can a. b. c. d.

give rise to every cell type in an organism’s body. produce an indefinite supply of a single cell type. produce a limited amount of a specific cell type. produce multiple cell types.

5. Plant meristems a. b. c. d.

are present only during development. contain stem cells. undergo meiosis. All of the choices are correct.

6. Pattern formation involves cells determining their position in the embryo. One mechanism that can accomplish this is a. b. c. d.

the loss of genetic material. alterations of chromosome structure. gradients of morphogens. changes in the cell cycle.

7. The process of nuclear reprogramming a. b. c. d.

is a normal part of pattern formation. reverses the changes that occur during differentiation. requires the introduction of new DNA. is not possible with mammalian cells.

A P P LY 1. What is the common theme in cell determination by induction or cytoplasmic determinants? a. The activation of transcription factors b. The activation of cell division c. A change in gene expression d. Both a and c are correct. 2. The process of reproductive cloning a. b. c. d.

shows that nuclear reprogramming is possible. is very efficient in mammals. always produces adult animals that are identical to the donor. Both a and b are correct.

3. Production of anterior–posterior and dorsal–ventral axes in the fruit fly Drosophila a. b. c. d.

both use gradients of mRNA. are conceptually similar but mechanistically different. use the exact same mechanisms. both use gradients of protein.

4. For pattern formation to occur, the cells in the developing embryo must a. b. c. d.

“know” their position in the embryo. be determined during the earliest divisions. differentiate as they are “born.” all be reprogrammed after each cell division.

5. The genes that encode the morphogen gradients in Drosophila were all identified in mutant screens. A mutation that removes the gradient necessary for the A/P morphogen gradient would be expected to a. b. c. d.

affect the larvae but not the adult. affect the adult but not the larvae. be lethal and lead to an abnormal embryo. produce replacement of one adult structure with another.

6. What would be the likely result of a mutation of the bcl-2 gene on the level of apoptosis? a. b. c. d.

No change A decrease in apoptosis An increase in apoptosis An initial decrease, followed by an increase in apoptosis

7. MADS box and Hox genes a. are found only in plants and animals, respectively. b. are found only in animals and plants, respectively. c. have similar roles in development in plants and animals, respectively. d. have similar roles in development in animals and plants, respectively.

SYNTHESIZE 1. The fate map for C. elegans (refer to figure 19.3) diagrams development of a multicellular organism from a single cell. Use this fate map to determine the number of cell divisions required to establish the population of cells that will become (a) the nervous system and (b) the gonads. 2. Carefully examine the C. elegans fate map in figure 19.3. Notice that some of the branchpoints (daughter cells) do not go on to produce more cells. What is the cellular mechanism underlying this pattern? 3. You have generated a set of mutant embryonic mouse cells. Predict the developmental consequences for each of the following mutations. a. Knockout mutation for N-cadherin b. Knockout mutation for integrin c. Deletion of the cytoplasmic domain of integrin 4. Assume you have the factors in hand necessary to reprogram an adult cell, and the factors necessary to induce differentiation to any cell type. How could these be used to replace a specific damaged tissue in a human patient? chapter

19 Cellular Mechanisms of Development 415

20

Part IV Evolution

CHAPTER

Genes Within Populations Chapter Contents 20.1

Genetic Variation and Evolution

20.2 Changes in Allele Frequency 20.3 Five Agents of Evolutionary Change 20.4 Quantifying Natural Selection 20.5 Reproductive Strategies 20.6 Natural Selection’s Role in Maintaining Variation 20.7 Selection Acting on Traits Affected by Multiple Genes 20.8 Experimental Studies of Natural Selection 20.9 Interactions Among Evolutionary Forces 20.10 The Limits of Selection

tamoncity/Shutterstock

Introduction

Visual Outline Genetic variation in populations acted on by Rosita So Image/Getty Images

Processes that cause evolutionary change include

Gene flow

Light

Dark

Average Tendency to Fly Toward Light

Mutation

11 10 9 8 7 6 5 4 3 2 1

Nonrandom mating

Genetic drift

Natural selection occurs when phenotypes differ in Fitness differences result from

0

2

4

6

8

10

12 14 16 18 20

Number of Generations

Survival

Mating success

Individuals in most populations vary greatly in phenotype, as the pigeons shown above illustrate; these differences are often the result of genetic differences among individuals. Often the particular characteristics of an individual have an important bearing on its survival, on its chances to reproduce, and on the success of its offspring. Evolution is driven by such factors, as the frequency of different alleles rises and falls in populations. These deceptively simple matters lie at the core of evolutionary biology, which is the topic of this chapter and of chapters 21–24.

Number of offspring per mating event

20.1 Genetic

Variation and Evolution

Learning Outcomes 1. Define evolution and population genetics. 2. Explain the importance and extent of variation within populations.

The term genetic variation refers to the different alleles of genes found within individuals of a population. Natural populations contain a wealth of such variation. In this chapter, we explore genetic variation in natural populations and consider the evolutionary forces that cause allele frequencies in natural populations to change. The word evolution is widely used in the natural and social sciences. It refers to how an entity—be it a social system, a gas, or a planet—changes through time. Although development of the modern concept of evolution in biology can be traced to Darwin’s landmark work, On the Origin of Species, the first five editions of his book never actually used the term. Rather, Darwin used the phrase “descent with modification.” Although many more complicated definitions have been proposed, Darwin’s words probably best capture the essence of biological evolution. Through time, species accumulate differences; as a result, descendants differ from their ancestors. In this way, new species arise from existing ones.

Many processes can lead to evolutionary change You have already learned about the development of Darwin’s ideas in chapter 1. Darwin was not the first to propose a theory of evolution. Rather, he followed a long line of earlier philosophers and naturalists who deduced that the many kinds of organisms around us were produced by a process of evolution. Unlike his predecessors, however, Darwin proposed natural selection as the mechanism of evolution. Natural selection produces evolutionary change when some individuals in a population possess certain inherited characteristics and then produce more surviving offspring than individuals lacking these characteristics. As a result, the population gradually comes to include more and more individuals with the advantageous characteristics. In this way, the population evolves and becomes better adapted to its local circumstances. One way to monitor how populations change through time is to look at changes in the frequencies of alleles of a gene from one generation to the next. Natural selection, by favoring individuals with certain alleles, can lead to change in such allele frequencies, but it is not the only process that can do so. Allele frequencies can also change when mutations occur repeatedly, changing one allele to another, and when migrants bring alleles into a population. In addition, the frequencies of alleles can change randomly as the result of chance events, particularly when populations are small. Often, natural selection overwhelms the effects of these other processes, but as you will see, this is not always the case. Evolution can result from any process that causes a change in the genetic composition of a population. We cannot talk about

Figure 20.1 Polymorphic variation. This natural population of lupines, Lupinus, exhibits considerable variation in flower color. Individual differences are inherited and passed on to offspring.

Rosita So Image/Getty Images

evolution, therefore, without also considering population genetics, the study of genetic variation within populations.

Populations contain ample genetic variation Genetic variation is required for evolution to occur. Consequently, biologists have always wanted to know how much genetic variation exists in natural populations. The ability to ask this question has been limited by the techniques available to analyze variation at different levels: proteins, genes, and now genomes. The story of genetic variation in natural populations is told using increasingly sophisticated tools for detecting differences. Initial approaches examined the most obvious differences, morphological variation. In many cases, such phenotypic variation is the result of underlying genetic differences. In such cases, natural populations usually show substantial genetic variation, as figure 20.1 illustrates.

Figure 20.2 Variation within a population of humans. Diverse Images/Universal Images Group/Getty Images chapter

20 Genes Within Populations 417

Other examples are closer to home. Consider all of the variation in a human population: skin, eye, and hair color; hair type; height; eye and nose shape; blood type; navel innie or outie; and many more. Most human variation is the result of underlying genetic differences (figure 20.2). The first approach to directly assay genetic variation within populations was the use of electrophoresis to separate proteins produced by alternative alleles of enzyme-encoding genes (see ­chapter 17). As each new DNA analysis tool was developed in the laboratory, it was quickly adapted for use with samples collected from natural populations. This led to, in order of increasing detail, examining restriction fragment length polymorphisms (RFLPs), sequencing specific genes, and, most recently, sequencing entire genomes. One of the most useful tools, both for genetic mapping and for the analysis of population-level variation, has been singlenucleotide polymorphisms (SNPs). These are defined as single-base differences between individuals that exist in the population at more than 1% (see chapter 24). An initial large-scale international effort identified several million SNPs in the human genome, a number we now know to be a vast underestimate. Such variation is also being assayed in many other species of interest. The results for most species are similar to those in humans: the closer you look, the more variation you see. One reason for the appeal of SNPs is that we have been able to automate the analysis of multiple samples. SNP data are now available for thousands of species, revealing the near ubiquity of genetic variation and providing tools for assessing patterns in human and natural populations. As genome sequencing moves into a phase where multiple genomes are being sequenced for many different species, a landscape of massive genetic variation is being revealed. Not surprisingly, the species with the most genomes sequenced is our own: by the time you read this, more than 100,000 human genomes will have been partially or entirely sequenced. As we saw in chapter 18, this revealed an enormous amount of genetic variation individuals will have around 3% of their genome varying from the reference genome. As well as extensive variation within human populations, these studies are also documenting substantial variation between populations. This kind of work is in progress looking at genomes from specifically Asian and African populations, and in one case a Dutch population. The early results from these forays into human genetic variation are significant and surprising. Not only do these populations have additional genetic variation not previously seen, but they also have entire genomic regions that are not represented in the reference genome which comes from individuals in North America. We have long known that African populations have greater genetic diversity than European and Asian populations. This is a result of human history: our species originated in Africa, and the populations there represent the “oldest” human genomes. A recent study that involved sequencing over 900 African genomes, from different regions of Africa, showed that taking all of these genomes into account, there was more than 300 Mb of DNA not found in the reference genome. It is becoming very clear that within a species, different populations have unique evolutionary trajectories and that substantial variation within a species is distributed across the species’ geographic range. Studies of genomic variation in natural populations are under way as well. More than 1000 genomes of the laboratory fruit fly, Drosophila melanogaster, have been completed. Comparisons of these genomes revealed extensive variation, although interestingly, 418 part IV  Evolution

some regions of the genome were more variable than others—just as with humans—and patterns of variation differed between the populations. In another study, 133 rhesus macaque (Macaca mulatta) genomes were sequenced, revealing substantial variation among these monkeys. Undoubtedly, the number of species for which many individuals are sequenced will skyrocket in the next few years, substantially enhancing our understanding of population variation.

Learning Outcomes Review 20.1

Evolution can be described as descent with modification. Variation is required for evolution to occur, and most populations contain extensive phenotypic and genotypic variation. Population genetics studies this variability. ■■

Why is genetic variation in a population necessary for evolution to occur?

20.2

Changes in Allele Frequency

Learning Outcomes 1. Explain the Hardy–Weinberg principle. 2. Describe the characteristics of a population that is in Hardy–Weinberg equilibrium. 3. Demonstrate how the operation of evolutionary processes can be detected.

Genetic variation within natural populations was a puzzle to Darwin and his contemporaries in the mid-1800s. Although Mendel performed his experiments during this same time period, his work was largely unknown. Selection, scientists then thought, should always favor an optimal form, and so tend to eliminate variation. Moreover, the theory of blending inheritance—in which offspring were expected to be phenotypically intermediate relative to their parents—was widely accepted. If blending inheritance were correct, then the effect of any new genetic variant would quickly be diluted to the point of disappearance in subsequent generations. For example, if a mutation made an individual 4 inches taller, its offspring, the result of a mating with an individual without that mutation, would be only 2 inches taller, and in the next generation’s offspring would be only 1 inch taller, and so on, until the effect completely disappeared.

The Hardy–Weinberg principle allows prediction of genotype frequencies Following the rediscovery of Mendel’s research, two people in 1908 solved the puzzle of why genetic variation persists—Godfrey H. Hardy, an English mathematician, and Wilhelm Weinberg, a German physician. These workers were initially confused about why, after many generations, a population didn’t come to be composed solely of individuals with the dominant phenotype. The conclusion they independently came to was that the original proportions of the genotypes in a population will remain constant from generation to generation, as long as the following assumptions are met:

1. No mutation takes place. 2. No genes are transferred to or from other sources (no immigration or emigration takes place). 3. Mating is random (individuals do not choose mates based on their phenotype or genotype). 4. The population size is very large. 5. No selection favoring one genotype over another occurs.

pened is p * p = p2 (figure 20.3). By the same reasoning, the probability that an individual will have two b alleles is q2. What about the probability that an individual will be a heterozygote? There are two ways this could happen: the individual could receive a B from its father and a b from its mother, or vice versa. The probability of the first case is p * q and the probability of the second case is q * p. Because the result in either case is that the individual is a heterozygote, the probability of that outcome is the sum of the two probabilities, or 2pq. So, to summarize, if a population is in Hardy–Weinberg equilibrium with allele frequencies of p and q, then the probability that an individual will have one of the three possible genotypes is p2 + 2pq + q2. You may recognize this as the binomial expansion:

If these assumptions are met, the genotypes’ proportions will not change, and the population is said to be in Hardy–Weinberg equilibrium.

The Hardy–Weinberg equation with two alleles: abinomial expansion In algebraic terms, the Hardy–Weinberg principle is written as an equation. Consider a population of 100 cats in which 84 are black and 16 are white. The frequencies of the two phenotypes would be 0.84 (or 84%) black and 0.16 (or 16%) white. Based on these phenotypic frequencies, can we deduce the underlying frequency of genotypes? If we assume that the white cats are homozygous recessive for an allele we designate as b, and the black cats are either homozygous dominant BB or heterozygous Bb, we can calculate the frequencies of the two alleles in the population from the proportion of black and white individuals, assuming that the population is in Hardy–Weinberg equilibrium. Let the letter p designate the frequency of the B allele and the letter q the frequency of the alternative allele. We call these allele frequencies. Because there are only two alleles, p plus q must always equal 1 (that is, the total population). In addition, we know that the sum of the three genotype frequencies must also equal 1. The probability that an individual will have two B alleles is simply the probability that each of its alleles is a B. The probability of two events happening independently is the product of the probability of each event; in this case, the probability that the individual received a B allele from its father is equal to the frequency of the allele in the population, p, and the probability the individual received a B allele from its mother is also p, so the probability that both hap-

(p + q)2 = p2 + 2pq + q2 Finally, we may use these probabilities to predict the distribution of genotypes in the population, again assuming that the population is in Hardy–Weinberg equilibrium. If the probability that any individual is a heterozygote is 2pq, then we would expect the proportion of heterozygous individuals in the population to be 2pq; similarly, the frequency of BB and bb homozygotes would be expected to be p2 and q2. Let us return to our example. Remember that 16% of the cats are white. If white is a recessive trait, then this means that such individuals must have the genotype bb. If the frequency of this genotype is q2 = 0.16 (the frequency of white cats), then q (the frequency of the b allele) = 0.4 (0.4 is the square root of 0.16). Because p + q = 1, therefore, p, the frequency of allele B, would be 1.0 – 0.4 = 0.6 (remember, the frequencies must add up to 1). We can now easily calculate the expected genotype frequencies: homozygous dominant BB cats would make up the p2 group, and the value of p2 = (0.6)2 = 0.36, or 36 homozygous dominant BB individuals in a population of 100 cats. The heterozygous cats have the Bb genotype and would have the frequency corresponding to 2pq, or (2 * 0.6 * 0.4) = 0.48, or 48 heterozygous Bb individuals.

Generation One

Phenotypes

84%

Generation Two B p = 0.60

b q = 0.40

B p = 0.60

BB p2 = 0.36

Bb pq = 0.24

b q = 0.40

Bb pq = 0.24

bb q2 = 0.16

16%

Eggs

Sperm Genotypes Frequency of genotype in population Frequency of gametes

BB

Bb

bb

0.36

0.48

0.16

0.36 + 0.24 = 0.60B

0.24 + 0.16 = 0.40b

p2 + 2 pq + q2 = 1

Figure 20.3 The Hardy–Weinberg equilibrium. In the absence of factors that alter them, the frequencies of gametes, genotypes, and phenotypes remain constant generation after generation.

Data analysis If all white cats died, what proportion of the kittens in the next generation would be white?

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20 Genes Within Populations 419

Using the Hardy–Weinberg equation to predict frequencies in subsequent generations The Hardy–Weinberg equation is another way of expressing the Punnett square described in chapter 12, with two alleles assigned frequencies, p and q. Figure 20.3 allows you to trace genetic reassortment during sexual reproduction and see how it affects the frequencies of the B and b alleles during the next generation. In constructing this diagram, we have assumed that the union of sperm and egg in these cats is random. The alleles are therefore represented in the next generation in proportion to their original occurrence. Each individual egg or sperm in each generation has a 0.6 chance of receiving a B allele (  p = 0.6) and a 0.4 chance of receiving a b allele (q = 0.4). In the next generation, therefore, the chance of combining two B alleles is p2, or 0.36 (that is, 0.6 * 0.6), and approximately 36% of the individuals in the population will continue to have the BB genotype. The frequency of bb individuals is q2 (0.4 * 0.4) and so will continue to be about 16%, and the frequency of Bb individuals will be 2pq (2 * 0.6 * 0.4), or on average, 48%. Phenotypically, if the population size remained at 100 cats, we would still see approximately 84 black individuals (with either BB or Bb genotypes) and 16 white individuals (with the bb genotype). Allele, genotype, and phenotype frequencies have remained unchanged from one generation to the next, despite the reshuffling of genes that occurs during meiosis and sexual reproduction. One important point to remember is that the terms “dominant” and “recessive” refer only to the phenotype of the heterozygote and in no way imply that one allele is selectively superior to another. For this reason, in Hardy-Weinberg equilibrium, when selection is not operating, allele frequencies are not expected to change regardless of whether one trait is dominant over another.

Hardy–Weinberg predictions can be applied to data to find evidence of evolutionary processes The lesson from the example of black and white cats is that if all five of the assumptions listed earlier in this section hold true, the allele and genotype frequencies will not change from one generation to the next. But in reality, most populations in nature will not fit all five assumptions. The primary utility of this method is to determine whether some evolutionary process or processes are operating in a population and, if so, to suggest hypotheses about what they may be. Suppose, for example, that the observed frequencies of the BB, bb, and Bb genotypes in a different population of cats were 0.6, 0.2, and 0.2, respectively. We can calculate the allele frequencies for B as follows: 60% (0.6) of the cats have two B alleles, 20% have one, and 20% have none. This means that the average number of B alleles per cat is 1.4 [(0.6 × 2) + (0.2 × 1) + (0.2 × 0) = 1.4]. Because each cat has two alleles for this gene, the frequency is 1.4/2.0 = 0.7. Similarly, you should be able to calculate that the frequency of the b allele = 0.3. If the population were in Hardy–Weinberg equilibrium, then, according to the equation earlier in this section, the frequency of the BB genotype would be 0.72 = 0.49, lower than it really is. Similarly, you can calculate that there are fewer heterozygotes and 420 part IV  Evolution

more bb homozygotes than expected; then clearly, the population is not in Hardy–­Weinberg equilibrium. What could cause such an excess of homozygotes and deficit of heterozygotes? A number of possibilities exist, including (1) natural selection favoring homozygotes over heterozygotes, (2) individuals choosing to mate with genetically similar individuals (because BB * BB and bb * bb matings always produce homozygous offspring, but only half of Bb * Bb produce heterozygous offspring, such mating patterns would lead to an excess of homozygotes), or (3) an influx of homozygous individuals from outside populations (or conversely, emigration of heterozygotes to other populations). By detecting a lack of Hardy–Weinberg equilibrium, we can generate potential hypotheses that we can then investigate directly. The operation of evolutionary processes can be detected in a second way. If all of the Hardy–Weinberg assumptions are met, then allele frequencies will stay the same from one generation to the next. Changes in allele frequencies between generations would indicate that one of the assumptions is not met. Suppose, for example, that the frequency of b was 0.53 in one generation and 0.61 in the next. Again, there are a number of possible explanations: for example, (1) selection favoring individuals with b over B, (2) immigration of b into the population or emigration of B out of the population, or (3) high rates of mutation that more commonly occur from B to b than vice versa. Another possibility is that the population is a small one, and that the change represents the random fluctuations that result because, simply by chance, some individuals pass on more of their genes than others. We will discuss how each of these processes is studied in the rest of the chapter.

Learning Outcomes Review 20.2

The Hardy–Weinberg principle states that in a large population with no selection and random mating, the proportion of alleles does not change through the generations. Finding that a population is not in Hardy–Weinberg equilibrium indicates that one or more evolutionary agents are operating. ■■

■■

If you know the genotype frequencies in a population, how can you determine whether the population is in Hardy– Weinberg equilibrium? What would you conclude if you found a population not in Hardy–Weinberg equilibrium? What would be your next step?

20.3

Five Agents of Evolutionary Change

Learning Outcomes 1. Define the five processes that can cause evolutionary change. 2. Explain how these processes can cause populations to deviate from Hardy–Weinberg equilibrium.

The five assumptions of the Hardy–Weinberg principle also indicate the five agents that can lead to evolutionary change in populations. They are mutation, gene flow, nonrandom mating, genetic drift in small populations, and the pressures of natural selection. Any one of these may bring about changes in allele or genotype proportions, causing the population to not be in HardyWeinberg equilibrium.

Mutation changes alleles Mutation from one allele to another can obviously change the proportions of particular alleles in a population. Mutation rates are generally so low that they have little effect on the Hardy–Weinberg proportions of common alleles. A typical gene mutates about once per 100,000 cell divisions. Because this rate is so low, other evolutionary processes are usually more important in determining how allele frequencies change. Nonetheless, mutation is the ultimate source of genetic variation and thus makes evolution possible (figure 20.4a). It is important to remember, however, that the likelihood that a particular mutation will occur is not affected by natural selection; that is, particular mutations do not occur more frequently in situations in which they would be favored by natural selection.

Gene flow occurs when alleles move between populations Gene flow is the movement of alleles from one population to another. It can be a powerful agent of change. Sometimes gene flow is obvious, as when an animal physically moves from one place to another. If the characteristics of the newly arrived individual differ from those of the animals already there, and if the newcomer is adapted well enough to the new area to survive and mate successfully, the genetic composition of the receiving population may be altered. Other important kinds of gene flow are not as obvious. These subtler movements include the drifting of gametes or the immature Mutation Mutagen

Gene Flow

DNA

stages of plants or marine animals from one place to another. ­Pollen, the male gamete of flowering plants, is often carried great distances by insects and other animals that visit flowers (figure 20.4b). Seeds may also blow in the wind or be carried by animals to new populations far from their place of origin. Consider two populations initially different in allele frequencies: in population 1, p = 0.2 and q = 0.8; in population 2, p = 0.8 and q = 0.2. Gene flow brings alleles into a population in proportion to their frequency in the source population. In this case, it would disproportionately bring in alleles that are rare in the recepient population because those alleles are common in the source. Thus, allele frequencies would change from generation to generation, and the populations would not be in Hardy–Weinberg equilibrium. Only when allele frequencies reach 0.5 for both alleles in both populations will equilibrium be attained (assuming that rates of gene flow are the same in both directions). This example also indicates that gene flow tends to homogenize allele frequencies among populations.

Nonrandom mating shifts genotype frequencies Individuals with certain genotypes sometimes mate with one another more commonly than would be expected on a random basis, a phenomenon known as nonrandom mating (­figure  20.4c). Assortative mating, in which phenotypically similar individuals mate, is a type of nonrandom mating that causes the frequencies of particular genotypes to differ greatly from those predicted by the Hardy–Weinberg principle. Assortative mating does not change the frequency of the individual alleles because it does not change the reproductive success of individuals, but rather changes with whom they mate. Because phenotypically similar individuals are likely to be genetically similar and thus are also more likely to produce offspring with two copies of the same allele, assortative mating will increase the proportion of homozygotes in the next generation.

Nonrandom Mating

Genetic Drift

Selection

Self-fertilization

C T

G

G C G A G

a. The ultimate source of

variation. Individual mutations occur so rarely that mutation alone usually does not change allele frequency much.

b. A very potent agent of

change. Individuals or gametes move from one population to another.

c. Inbreeding, of which mating

with one's self is the most extreme form, is a common type of nonrandom mating. It does not alter allele frequency, but reduces the proportion of heterozygotes.

d. Statistical accidents. The

random fluctuation in allele frequencies increases as population size decreases.

e. The only agent that

produces adaptive evolutionary changes.

Figure 20.4 Five agents of evolutionary change. a. Mutation. b. Gene flow. c. Nonrandom mating. d. Genetic drift. e. Selection. chapter

20 Genes Within Populations 421

Inbreeding results when closely related individuals mate with each other. Because relatives are genetically similar, inbreeding will have the same result as assortative mating and increase the proportion of homozygotes. In fact, because relatives tend to be phenotypically similar, assortative mating can lead to inbreeding. By contrast, disassortative mating, in which pheno­typically different individuals mate, produces an excess of heterozygotes.

Genetic drift may alter allele frequencies in small populations In small populations, frequencies of particular alleles may change drastically by chance alone. Such changes in allele frequencies occur randomly, as if the frequencies were drifting from their values. These changes are thus known as genetic drift (figure 20.4d). For this reason, a population must be large to be in Hardy–Weinberg equilibrium: although drift occurs in all populations, the smaller the population, the greater the change is in allele frequencies from generation to generation as a result of genetic drift. If the gametes of only a few individuals form the next generation, the alleles they carry may by chance not be representative of the parent population from which they were drawn, as illustrated in figure 20.5. In this example, a small number of individuals are removed from a bottle. By chance, most of the individuals removed are green, so the new population has a much higher population of green individuals than the parent generation had. Suppose, for example, that from a population in which B and b are equally represented (that is, p = q = 0.5), four individuals formed the next generation, and that by chance they were two Bb heterozygotes and two BB homozygotes—that is, the allele frequencies in the next generation would be p = 0.75 and q = 0.25. In fact, if you were to replicate this experiment 1000 times, each time randomly­drawing four individuals from the parental population, then in about 8 of the 1000 experiments, one of the two alleles would be missing entirely. This result leads to an important conclusion: genetic drift can lead to the loss of alleles in isolated populations. Alleles that initially are uncommon are particularly vulnerable (figure 20.5). A set of small populations that are isolated from one another may come to differ strongly as a result of genetic drift, even if the forces of natural selection are the same for all. Because of genetic drift, sometimes harmful alleles may increase in frequency in small populations, despite selective disadvantage, and favorable alleles may be lost even though they are selectively advantageous. Although genetic drift occurs in any population, its effect is inversely related to population size. Thus, it is particularly likely in populations that were founded by a few individuals or in which the population was reduced to a very small number at some time in the past. Such founder effects or bottlenecks are particularly clear examples of genetic drift.

The founder effect Sometimes one or a few individuals disperse and become the founders of a new, isolated population at some distance from their place of origin. These pioneers are not likely to carry all the alleles 422 part IV  Evolution

Parent population

Bottleneck (drastic reduction in population)

Surviving individuals

Next generation

Figure 20.5 Genetic drift: a bottleneck effect. The

parent population contains roughly equal numbers of green and yellow individuals and a small number of red individuals. By chance, the few remaining individuals that contribute to the next generation are mostly green and the red allele has disappeared entirely. The bottleneck occurs because so few individuals form the next generation, as might happen after an epidemic or a catastrophic storm.

?

Inquiry question Why are rare alleles particularly likely to be lost in a population bottleneck?

present in the source population. Thus, some alleles may be lost from the new population, and others may change drastically in frequency. In some cases, previously rare alleles in the source population may be a significant fraction of the new population’s genetic endowment. This phenomenon, in which the allele frequencies of the founding individuals are different from the allele frequencies in the population from which they came, is called the founder effect. Founder effects are not rare in nature. Many ­self-­pollinating plants start new populations from a single seed. Founder effects have been particularly important in the evolution of organisms on distant oceanic islands, such as the Hawaiian and Galaˊpagos Islands. Most of the organisms in such areas probably derive from one or a few initial founders. Although rare, such events are occasionally observed, such as when a mass of vegetation carrying several iguanas washed up on the shore of the Caribbean island of Anguilla in 1996, leading to the establishment of a population that still occurs there to this day. In a similar way, isolated human populations begun by relatively few individuals are often dominated by genetic features characteristic of their founders. Amish populations in the United States, for example, were established from relatively few individuals and have unusually high frequencies of a number of conditions, such as polydactylism (the presence of a sixth finger).

The bottleneck effect Even if organisms do not move from place to place, occasionally their populations may be drastically reduced in size. This may result from flooding, drought, epidemic disease, and other natural forces, or from changes in the environment. Just as we have seen with founder effects, the few surviving individuals may by chance have allele frequencies different from those in the entire

population, and some alleles may be lost entirely (natural selection may also be responsible for change in allele frequencies, but here we are referring to random changes resulting from small population size). The resulting alterations and loss of genetic variability have been termed the bottleneck effect. The genetic variation of some living species appears to be severely depleted, probably as the result of a bottleneck effect in the past. For example, the northern elephant seal, which breeds on the western coast of North America and nearby islands, was nearly hunted to extinction in the 19th century and was reduced to a single population containing perhaps no more than 20 individuals on the island of Guadalupe off the coast of Baja California (figure 20.6). As a result of this bottleneck, the species has lost almost all of its genetic variation, even though the seal populations have rebounded and now number in the tens of thousands and breed in locations as far north as near San Francisco. Any time a population becomes drastically reduced in numbers, such as in endangered species, the bottleneck effect is a potential problem. Even if population size rebounds, the lack of variability may mean that the species remains vulnerable to extinction—a topic to which we will return in chapter 58.

Selection favors some phenotypes over others As Darwin pointed out, some individuals leave behind more progeny than others, and the rate at which they do so is affected by their phenotype. We describe the results of this process as selection, when individuals with one phenotype leave, on average, more surviving offspring in the next generation than individuals with an alternative phenotype (see figure 20.4e). In artificial selection, a breeder selects for the desired characteristics. In natural selection, environmental conditions determine which individuals in a population produce the most offspring.

UNITED S TAT E S

Evolution by natural selection occurs when the following conditions are met: 1. Phenotypic variation must exist among individuals in a population. Natural selection works by favoring individuals with some traits over individuals with alternative traits. If no variation exists, natural selection cannot operate. 2. Variation among individuals must result in differences in the number of offspring surviving in the next generation. This is natural selection. Because of their phenotype or behavior, some individuals are more successful than others in producing offspring. Although many traits are phenotypically variable, individuals exhibiting variation do not always differ in survival and reproductive success. 3. Phenotypic variation must have a genetic basis. For natural selection (see item 2) to result in evolutionary change, the selected differences must have a genetic basis. Not all variation has a genetic basis—even genetically identical individuals may be phenotypically quite distinctive if they grow up in different environments. Such environmental effects are common in nature. In many turtles, for example, individuals that hatch from eggs laid in moist soil are heavier, with longer and wider shells, than individuals from nests in drier areas. When phenotypically different individuals do not differ genetically, differences in the number of their offspring will not alter the genetic composition of the population in the next generation, and thus, no evolutionary change will have occurred. It is important to remember that natural selection and evolution are not the same—the two concepts often are incorrectly

population in 1890, reduced to inhabiting Guadalupe only current population

Figure 20.6 Bottleneck effect: case study. Because northern elephant seals,

Mirounga angustirostris, live in very cold waters, they have thick layers of fat, for which they were hunted nearly to extinction late in the 19th century. At the low point, only one population remained, on Guadalupe Island, with perhaps as few as 20 individuals; during this time, genetic variation was lost. Since being protected, the species has reclaimed most of its original range and now numbers in the tens of thousands, but genetic variation will recover only slowly over time as mutations accumulate. Guadalupe MEXICO

chapter

20 Genes Within Populations 423

equated. Natural selection is a process, whereas evolution is the historical record, or outcome, of change through time. Natural selection (the process) can lead to evolution (the outcome), but natural selection is only one of several processes that can result in evolutionary change. Moreover, natural selection can occur without producing evolutionary change; only if variation is genetically based will natural selection lead to evolution.

Selection to avoid predators A common result of evolution driven by natural selection is that populations become better adapted to their environment. Many of the most dramatic documented instances of adaptation involve genetic changes that decrease the probability of capture by a predator. The caterpillar larvae of the common sulphur butterfly Colias eurytheme usually exhibit a pale green color, providing excellent camouflage against the alfalfa plants on which they feed. An alternative bright yellow color morph is reduced to very low frequency because this color renders the larvae highly visible on the food plant, making it easier for bird predators to see them (see figure 20.4e). One of the most dramatic examples of background matching involves ancient lava flows in the deserts of the American Southwest. In these areas, the black rock formations produced when the lava cooled contrast starkly with the surrounding bright glare of the desert sand. Populations of many species of animals occurring on these rocks—including lizards, rodents, and a variety of insects—are dark in color, whereas sand-dwelling populations in surrounding areas are much lighter (figure 20.7). Predation is the likely cause for these differences­in color. Laboratory studies have confirmed that predatory birds such as owls are adept at picking out individuals occurring on backgrounds to which they are not adapted.

Light coat color pocket mouse is vulnerable on lava rock

Light coat color favored by natural selection because it matches sand color

Dark coat color favored by natural selection because it matches black lava rock

Figure 20.7 Pocket mice from the Tularosa Basin of New Mexico whose color matches their background. Black lava formations are surrounded by desert, and selection favors coat color in pocket mice that matches their surroundings. Genetic studies indicate that the differences in coat color are the result of small differences in the DNA of alleles of a single gene. 424 part IV  Evolution

Selection to match climatic conditions Many studies of selection have focused on genes encoding enzymes, because in such cases the investigator can directly assess the consequences to the organism of changes in the frequency of alternative enzyme alleles. Often investigators find that enzyme allele frequencies vary with latitude, so that one allele is more common in northern populations, but is progressively less common at more southern locations. A superb example is seen in studies of a fish, the mummichog (Fundulus heteroclitus), which ranges along the eastern coast of North America. In this fish, geographic variation occurs in allele frequencies for the gene that produces the enzyme lactate dehydrogenase, which catalyzes the conversion of pyruvate to lactate (see section 7.8). Biochemical studies show that the enzymes formed by these alleles function differently at different temperatures, thus explaining their geographic distributions. The form of the enzyme more frequent in the north is a better catalyst at low temperatures than is the enzyme from the south. Moreover, studies indicate that at low temperatures, individuals with the northern allele swim faster, and presumably survive better, than individuals with the alternative allele.

Selection for pesticide and microbial resistance A particularly clear example of selection in natural populations is provided by studies of pesticide resistance in insects. The widespread use of insecticides has led to the rapid evolution of resistance in more than 500 pest species. The cost of this evolution, in terms of crop losses and increased pesticide use, has been estimated at $3–8 billion per year. In the housefly, the resistance allele of the pen gene decreases the uptake of insecticide, whereas alleles of the kdr and dld-r genes decrease the number of target sites, thus decreasing the binding ability of the insecticide (figure 20.8). Other alleles enhance the ability of the insects’ enzymes to identify and detoxify insecticide molecules. Single genes are also responsible for resistance in other organisms. For example, Norway rats are normally susceptible to the pesticide warfarin, which diminishes the clotting ability of the rat’s blood and leads to fatal hemorrhaging. However, a resistance allele of the VKORC1 gene reduces the ability of warfarin to bind to its target enzyme and thus renders it ineffective. Selection imposed by humans has also led to the evolution of resistance to antibiotics in many disease-causing pathogens. For example, Staphylococcus aureus, which causes staph infections, was initially treated by penicillin, which latched onto S. aureus, degrading cell walls and causing the death of the bacteria. However, within four years of mass-production of the drug, evolutionary change in S. aureus modified an enzyme, penicillinase, so that it would attack penicillin, making the drug unable to attach to S. aureus and thus rendering it ineffective. Since that time, several other drugs have been developed to attack the microbe, and each time resistance has evolved. As a result, staph infections have re-emerged as a major health threat. The speed by which adaptation occurs may be surprising, but remember that microbes have enormous population sizes. Even though mutation rates are low, the vast size of these populations guarantees a steady supply of new mutations for natural

Pesticide molecule

Target site

Resistant target site

Insect cell membrane

a. Insect cells with resistance allele at pen gene: decreased uptake of the pesticide.

Target site

b. Insect cells with resistance allele at kdr gene:

decreased number of target sites for the pesticide.

Figure 20.8 Selection for pesticide resistance. 

Resistance alleles of genes such as pen and kdr allow insects to be more resistant to pesticides. Insects that possess these resistance alleles have become more common through selection.

selection to utilize. The effect of such evolution on human health is enormous. In the United States alone, 2 million people each year become ill due to antibiotic-resistant bacteria, and 23,000 die from such infections.

Learning Outcomes Review 20.3

Five factors can bring about deviation from the predicted Hardy–Weinberg genotype frequencies. Of these, only selection regularly produces adaptive evolutionary change, but the genetic constitution of populations, and thus the course of evolution, can also be affected by mutation, gene flow, nonrandom mating, and genetic drift. ■■

How does each of these processes cause populations to vary from Hardy–Weinberg equilibrium?

20.4

Quantifying Natural Selection

Learning Outcomes 1. Define evolutionary fitness. 2. Explain the different components of fitness. 3. Demonstrate how the success of different phenotypes can be compared by calculating their relative fitness.

Selection occurs when individuals with one phenotype leave more surviving offspring in the next generation than individuals with an alternative phenotype. Evolutionary biologists quantify reproductive success as fitness, the number of surviving offspring left in the next generation. Fitness is a relative concept; the most fit phenotype is simply the one that produces, on average, the greatest number of offspring.

A phenotype with greater fitness usually increases in frequency Suppose, for example, that in a population of toads, two phenotypes exist: green and brown. Suppose, further, that green toads leave, on average, 4.0 offspring in the next generation, but brown toads leave only 2.5. By custom, the most fit phenotype is assigned a fitness value of 1.0, and other phenotypes are expressed as relative proportions. In this case, the fitness of the green phenotype would be 4.0/4.0 = 1.000, and the fitness of the brown phenotype would be 2.5/4.0 = 0.625. The difference in fitness would therefore be 1.000 – 0.625 = 0.375. A dif­ference in fitness of 0.375 is quite large; natural selection in this case strongly favors the green phenotype. If differences in color have a genetic basis, then we would expect evolutionary change to occur; the frequency of green toads should be substantially greater in the next generation. Further, if the fitness of two phenotypes remained unchanged, we would expect alleles for the brown phenotype eventually to disappear from the population.

?

Inquiry question Why might the frequency of green toads not increase in the next generation, even if color differences have a genetic basis?

Fitness may consist of many components Although selection is often characterized as “survival of the ­fittest,” differences in survival are only one component of fitness. Even if no differences in survival occur, selection may operate if some individuals are more successful than others in attracting mates. In many territorial animal species, for example, large males mate with many females, and small males rarely get to mate. Selection with respect to mating success is termed sexual selection; we describe this topic more fully in the next section. In addition, the number of offspring produced per reproductive event is also important. Large female frogs and fish lay more eggs than do smaller females, and thus they may leave more offspring in the next generation. Fitness is therefore a combination of survival, mating success, and number of offspring per mating. Selection favors phenotypes with the greatest fitness, but predicting fitness from a single component can be tricky because traits favored for one component of fitness may be at a disadvantage for others. As an example, in water striders, larger females lay more eggs per day (figure 20.9). Thus, natural selection at this stage favors large size. However, larger females also die at a younger age and thus have fewer opportunities chapter

20 Genes Within Populations 425

8 Number of Eggs Laid per Day

6 4 2

200 Number of Eggs Laid During Lifetime

Life Span of Adult Female (days)

50 40 30 20 10 0

0 12

13

14

15

12

16

Length of Adult Female Water Strider (mm)

13

14

15

16

Length of Adult Female Water Strider (mm)

150 100 50 0 12

13

14

15

16

Length of Adult Female Water Strider (mm)

Figure 20.9 Body size and egg-laying in water striders. Larger female water striders lay more eggs per day (left panel), but also survive for a shorter period of time (center panel). As a result, intermediate-sized females produce the most offspring over the course of their entire lives and thus have the highest fitness (right panel).

?

Inquiry question What evolutionary change in body size might you expect? If the number of eggs laid per day was not affected by body size, would your prediction change?

Data analysis Assuming that the values on the x-axis represent the final body size of different animals and that the y-axis represents egg-laying rate and survival once they reach that size, how many eggs would you expect a 12-mm water strider to lay? And a 15-mm strider?

to reproduce than smaller females. Overall, the two opposing directions of selection cancel each other out, and the intermediate-sized females leave the most offspring in the next generation.

Learning Outcomes Review 20.4

Fitness is defined by an organism’s reproductive success relative to other members of its population. This success is determined by how long it survives, how often it mates, and how many offspring it produces per mating. Relative fitness assigns numerical values to different phenotypes relative to the most fit phenotype. ■■

Is one of these factors always the most important in determining reproductive success? Explain.

20.5

Reproductive Strategies

Learning Outcomes 1. Explain parental investment and the prediction it makes about mate choice. 2. Understand the difference between intra- and intersexual selection. 3. Describe how sexual selection leads to the evolution of secondary sexual characteristics.

Males and females have the common goal of improving the quantity and quality of offspring they produce, but usually differ in the way 426 part IV  Evolution

they attempt to maximize fitness. Such a difference in reproductive behavior is clearly seen in mate choice. Darwin was the first to observe that females often do not mate with the first male they encounter, but instead seem to evaluate a male’s quality and then decide whether to mate. Peahens prefer to mate with peacocks that have more eye­spots on their elaborate tail feathers (figure 20.10b, c). Similarly, female frogs prefer to mate with males having more acoustically complex, and thus attractive, calls. This behavior, called mate choice, is well known in many invertebrate and vertebrate species.

The sexes often have different reproductive strategies Males are selective in choosing a mate much less frequently than females. Why should this be? Many of the differences in reproductive strategies between the sexes can be understood by comparing the parental investment made by males and females. Parental investment refers to the energy and time each sex “invests” in producing and rearing offspring; it is, in effect, an estimate of the energy expended by males and females in each reproductive event. Numerous studies have shown that females generally have a higher parental investment. One reason is that eggs are much larger than sperm—195,000 times larger in humans! Eggs contain proteins and lipids in the yolk and other nutrients for the developing embryo, but sperm are little more than mobile DNA packages. In some groups of animals (mammals, for example), females are responsible for gestation and lactation, costly reproductive functions only they can carry out. The consequence of such inequalities in reproductive investment is that the sexes face very different selective pressures. Because any single reproductive event is relatively inexpensive for males, they can best increase their fitness by mating with as many females as possible. This is because male fitness is likely not

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Figure 20.10 Products of sexual selection. In bird species

such as a. the peacock, Pavo cristatus, and b. the raggiana bird of paradise, Paradisaea raggiana, males use their much longer tail feathers in courtship displays. c. Female peahens prefer to mate with males having greater numbers of eyespots in their tail feathers. (a) Carole_

R/Flickr Flash/Getty Images; (b) Bruce Beehler/Science Source

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limited by the amount of sperm they can produce. By contrast, each reproductive event for females is much more costly, and the number of eggs that can be produced often limits reproductive success. For this reason, a female should be choosy, trying to pick the male that can provide the greatest benefit to her offspring and thus improve her fitness. These conclusions hold only when female reproductive investment is much greater than that of males. In species with biparental care, males may contribute equally to the cost of raising young; in this case, the degree of mate choice should be more equal between the sexes. In some cases, male investment exceeds that of females. For example, male Mormon crickets transfer a protein-containing packet (a spermatophore) to females during mating. Almost 30% of a male’s body weight is made up by the spermatophore, which provides nutrition for the female and helps her develop her eggs. As we might expect from our model of mate choice, in this case it is the females that compete with one another for access to males, which are the choosy sex. I­ ndeed, males are quite selective, favoring heavier females. Heavier females have more eggs; thus, males that choose larger females leave more offspring (figure 20.11). Males care for eggs and developing young in many species, including seahorses and a number of birds and insects. As with Mormon crickets, these males are often choosy, and females compete for mates.

Sexual selection occurs through mate competition and mate choice The reproductive success of an individual is determined by how long the individual lives, how frequently it mates, and how many offspring it produces per mating. The second of these factors, competition for mates, is termed sexual selection. Some people

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Inquiry question Why do females prefer males with more spots?

Data analysis Suppose a male had 155 eyespots. How many females would you expect him to mate with? If you had multiple males with 155 eyespots, do you think they all would mate with the same number of females?

consider sexual selection to be distinctive from natural selection, but others see it as a subset of natural selection, just one of the many factors affecting an organism’s fitness. Sexual selection involves both intrasexual selection, or competitive interactions between members of one sex (“the power to conquer other males in battle,” as Darwin put it), and intersexual selection, which is another name for mate choice (“the power to charm”). Sexual selection leads to the evolution of structures used in combat with other males, such as a deer’s antlers and a ram’s horns, as well as ornaments used to “persuade” members of the opposite sex to mate, such as long tail feathers and bright plumage (see figure 20.10a, b). These traits are called secondary sexual characteristics. Selection strongly favors any trait that confers greater ability in mate competition. Larger body size is a great advantage if dominance is important, as it is in territorial species. Males may thus be considerably larger than females. Such differences between the sexes are referred to as sexual dimorphism. In other species, structures used for fighting, such as horns, antlers, and large canine teeth, have evolved to be larger in males because of the advantage they give in intrasexual competition. Sometimes sperm competition occurs between the sperm of different males if females mate with multiple males. This type of competition, which occurs after mating, has selected for features that increase the probability that a male’s sperm will fertilize the eggs. Such features include sperm-transfer organs designed to remove the sperm of a prior mating, large testes to produce more sperm per mating, and sperm that hook themselves together to swim more rapidly. chapter

20 Genes Within Populations 427

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Figure 20.11 The advantage of male mate choice. Male Mormon crickets, Anabrus simplex,

choose heavier females as mates, and larger females have more eggs. Thus, male mate selection increases fitness.

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Inquiry question Is there a benefit to females for mating with large males?

Intrasexual selection In many species, individuals of one sex—usually males—compete with one another for the opportunity to mate. Competition among males can occur for a territory in which females feed or bear young. Males may also directly compete for the females themselves. A few successful males may engage in an inordinate number of matings, whereas most males do not mate at all. For example, elephant seal males control territories on breeding beaches and a few dominant males do most of the breeding (figure 20.12). On one beach, for example, eight males impregnated 348 females, whereas the remaining males mated rarely, if at all.

Indirect benefits of mate choice. In many species, however, males provide no direct benefits of any kind to females. In such cases, it is not intuitively obvious what females have to gain by being “choosy.” Moreover, what could be the possible benefit of choosing a male with an extremely long tail or a complex song? One possible indirect benefit of mate choice is that it could lead to higher-quality offspring. A number of theories have been proposed as to how this might work. One idea is that females choose the male that is the healthiest or oldest. Large males, for example, have probably been successful at living long, acquiring a lot of food, and resisting parasites and disease. In other species, features other than size may indicate a male’s condition. In guppies and some birds, the brightness of a male’s color reflects the quality of his diet and overall health. Females may gain two benefits from mating with the healthiest males. First, healthy males are less likely to be carrying diseases, which might be transmitted to the female during mating; this would be a direct benefit. Second, to the extent that the males’ success in living long and prospering is the result of his genetic makeup, the female will be ensuring that her offspring receive good genes from their father, an indirect benefit. Several experimental studies in fish and moths have examined whether female mate choice leads to greater reproductive success. In these experiments, females in one group were allowed to choose the males with which they would mate, whereas males were randomly mated to a different group of females. Offspring of females that chose their mates were more vigorous and survived better than offspring from females given no choice, which suggests that females preferred males with a better genetic makeup. A variant of this theory goes one step further. In some cases, females prefer mates with traits that appear to be detrimental to survival (see figure 20.10c). The long tail of the

Intersexual selection Intersexual selection concerns the active choice of a mate. Mate choice has both direct and indirect benefits. Direct benefits of mate choice.  In some cases, the female benefits directly from her choice of mates. If males help raise offspring, females benefit by choosing the male that can provide the best care—the better the parent, the more offspring she is likely to rear. In other species, males provide no care, but maintain territories that provide food, nesting sites, and predator refuges. In red deer, males that hold territories with the highest-quality grasses mate with the most females. In this case, there is a direct benefit of a female mating with such a territory owner: she feeds with little disturbance on quality food. 428 part IV  Evolution

Figure 20.12 Male northern elephant seals (Mirounga angustirostris) compete for mates. Male northern elephant seals

fight with one another for possession of territories. Only the largest males can hold territories, which contain many females. ©Cathy & Gordon ILLG

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Figure 20.13 Male Túngara frog, Physalaemus pustulosus, calling. Female frogs of several species in the genus Physalaemus prefer males that include a “chuck” in their call. However, only males of the Túngara frog a. produce such calls b. males of other species do not c. W. Perry Conway/Getty Images

peacock is a hindrance in flying and makes males more vulnerable to predators. Why should females prefer males with such traits? The handicap hypothesis states that only genetically superior mates can survive with such a handicap. By choosing a male with the largest handicap, the female is ensuring that her offspring will receive these quality genes. Of course, the male offspring will also inherit the genes for the handicap. For this reason, evolutionary biologists are still debating the merit of this hypothesis. Alternative theories about the evolution of mate choice.  Some courtship displays appear to have evolved from a predisposition in the female’s sensory system to respond to certain stimuli. For example, females may be better able to detect particular colors or sounds at a certain frequency, and thus be attracted to such signals. Sensory exploitation involves the evolution in males of a signal that “exploits” these preexisting biases. For example, if females are particularly adept at detecting red objects, then red coloration may evolve in males as part of a courtship display. To understand the evolution of courtship calls, consider the vocalizations of the Tu´ngara frog (figure 20.13). Unlike related species, males include a short burst of sound, termed a “chuck,” at the end of their calls. Recent research suggests that not only are females of this species particularly attracted to calls of this sort, but so are females of related species, even though males of these species do not produce “chucks.” This pattern suggests that the preference evolved for some reason in the ancestor of all of the species, and then subsequently led to the evolution of the chuck only in males of the Tu´ngara frog. Why males of the other species haven’t evolved to produce chucks is a good question. The opportunity for sensory exploitation may be widespread. For example, researchers conducted an experiment to see if birds had latent preferences for particular stimuli using a bird common in the pet trade, the zebra finch. To conduct the experiment, the researchers glued long feathers vertically to the tops of the heads of male birds (figure 20.14). The result was that the males had very tall crests, completely unlike anything seen in nature. They then presented males with different colored crests to females to see if the females had a preference for one color over another. Surprisingly, females were strongly attracted to males

with white crests, but not green or red ones. Why this might be is not at all clear; it suggests there is something in the zebra finch’s neural hardwiring that for some reason finds white much more attractive than green or red. Regardless of the cause of the preference, this finding suggests that if a mutation arose that caused a male to have a white crest on his head, the mutation would bestow a great advantage and would quickly spread through the population. Perhaps there are many such latent preferences in species—preferences we might never be able to predict and that might be hard to explain. But if appropriate variation arises in a population, females may prefer it. This is a fascinating area of research that is just in its infancy, and will require coupling studies of neurophysiology and behavior to understand how and why such mate choice preferences evolve.

Figure 20.14 Male zebra finches with artificial crests. 

Researchers glued different colored feathers on the heads of male zebra finches to see if females exhibited a preference for one color over another. chapter

20 Genes Within Populations 429

Learning Outcomes Review 20.5

The sex that invests more in reproduction (parental investment) tends to exhibit mate choice. Females or males can be selective, depending on the energy and time they devote to parental care. Sexual selection refers to differences among individuals of the same sex in mating success. Intrasexual selection results when individuals of the same sex compete for matings, whereas intersexual selection occurs when members of one sex choose which individuals of the other sex with whom to mate. Sexual selection governs evolution of secondary sex characteristics in that mates are chosen on the basis of phenotype and competitive success. ■■

Pipefish males incubate young in a brood pouch, a process that takes two weeks, during which time males cannot accept further eggs. Which sex would you expect to show mate choice? Why?

20.6

Natural Selection’s Role in Maintaining Variation

Learning Outcomes 1. Define frequency-dependent selection, oscillating selection, and heterozygote advantage. 2. Explain how these processes affect the amount of genetic variation in a population.

Natural selection has been discussed as a process that removes variation from a population by favoring one allele over others at a gene locus. However, in some circumstances, selection can do exactly the opposite and actually maintain population variation.

Frequency-dependent selection may favor either rare or common phenotypes In some circumstances, the fitness of a phenotype depends on its frequency within the population, a phenomenon termed frequency-dependent selection. This type of selection favors certain phenotypes depending on how commonly or uncommonly they occur.

Negative frequency-dependent selection In negative frequency-dependent selection, rare phenotypes are favored by selection—thus, fitness has a negative relationship with 430 part IV  Evolution

SCIENTIFIC THINKING Question: Does negative frequency-dependent selection maintain variation in a population? Hypothesis: Fish may disproportionately capture water boatmen (a type of aquatic insect) with the most common color. Experiment: Place predatory fish in different aquaria with the different frequencies of the color types in each aquarium. Result: Fish prey disproportionately on the common color in each aquarium. The rare color in each aquarium generally survives best. 100

Percent of Color Type Taken by Fish Predators

A great variety of other hypotheses have been proposed to explain the evolution of mating preferences. Many of these hypotheses may be correct in some circumstances, but none seems capable of explaining all of the variation in mating behavior in the animal world. This is an area of vibrant research, with new discoveries appearing regularly.

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Figure 20.15 Frequency-dependent selection.

Data analysis How can this experiment distinguish between frequency-dependent and directional selection? phenotype frequency, hence the term negative frequency-dependent. Assuming a genetic basis for phenotypic variation, such selection will have the effect of making rare alleles more common, thus maintaining variation. Negative frequency-dependent selection can occur for many reasons. For example, it is well known that animals or people searching for something form a “search image.” That is, they become particularly adept at picking out certain objects. Consequently, predators may form a search image for common prey phenotypes. Rare forms may thus be preyed upon less frequently. An example is fish predation on an insect, the water boatman, which occurs in three different colors. Experiments indicate that each of the color types is preyed upon disproportionately when it is the most common one; fish eat more of the common-colored insects than would occur by chance alone (figure 20.15). Another cause of negative frequency dependence is resource competition. If genotypes differ in their resource requirements, as occurs in many plants, then the rarer geno­type will have fewer competitors. When the different resource types are equally abundant, the rarer genotype will be at an advantage relative to the more common genotype.

change in frequencies themselves that leads to the changes in fitness of the different phenotypes.

In some cases, heterozygotes may exhibit greater fitness than homozygotes

Figure 20.16 Positive frequency-dependent selection.  In some cases, rare individuals stand out from the rest and draw the attention of predators; thus, in these cases, common phenotypes have the advantage (positive frequency-dependent selection).

If heterozygotes are favored over homozygotes, then natural selection actually tends to maintain variation in the population. This heterozygote advantage favors individuals with copies of both alleles, and thus works to maintain both alleles in the population. Some evolutionary biologists think that heterozygote advantage is pervasive and can explain the high levels of genetic variation observed in natural populations. Others, however, consider it to be relatively rare. The best-documented example of heterozygote advantage is sickle cell anemia, a hereditary disease affecting hemoglobin in humans. Individuals with sickle cell anemia exhibit symptoms of severe anemia and abnormal red blood cells that are irregular in shape, with a great number of long, sickle-shaped cells (­figure 20.17). Chapter 13 discusses why the sickle cell mutation (S) causes red blood cells to sickle. The average incidence of the S allele in central African populations is about 0.12, far higher than that found among Americans of African descent. From the Hardy–Weinberg principle, you can

Positive frequency-dependent selection Positive frequency-dependent selection has the opposite effect; by favoring common forms, it tends to eliminate variation from a population. For example, predators don’t always select common individuals. In some cases, “oddballs” stand out from the rest and attract attention (figure 20.16). The strength of selection should change through time as a result of frequency-dependent selection. In negative frequency-­ dependent selection, rare genotypes should become increasingly common, and their selective advantage will decrease correspondingly. Conversely, in positive frequency dependence, the rarer a geno­ type becomes, the greater the chance it will be selected against.

Normal red blood cells Sickled red blood cells

In oscillating selection, the favored phenotype changes as the environment changes In some cases, selection favors one phenotype at one time and another phenotype at another time, a phenomenon called oscillating selection. If selection repeatedly oscillates in this fashion, the effect will be to maintain genetic variation in the population. One example, discussed in chapter 21, concerns the medium ground finch of the Galaˊpagos Islands. In times of drought, the supply of small, soft seeds is depleted, but there are still enough large seeds around. Consequently, birds with big bills are favored. However, when wet conditions return, the ensuing abundance of small seeds favors birds with smaller bills. Oscillating selection and frequency-dependent selection are similar because in both cases the form of selection changes through time. But it is important to recognize that they are not the same: in oscillating selection, the fitness of a phenotype does not depend on its frequency; rather, environmental changes lead to the oscillation in selection. In contrast, in ­frequency-dependent selection, it is the

Sickle cell allele in Africa 1–5% 5–10% 10–20% Geographic distribution of P. falciparum malaria

Figure 20.17 Frequency of sickle cell allele and distribution of Plasmodium falciparum malaria. The red

blood cells of people homozygous for the sickle cell allele collapse into sickled shapes when the oxygen level in the blood is low. The distribution of the sickle cell allele in Africa coincides closely with that of P. falciparum malaria. chapter

20 Genes Within Populations 431

calculate that 1 in 5 central African individuals is heterozygous at the S allele, and 1 in 100 is homozygous and develops the fatal form of the disorder. People who are homozygous for the sickle cell allele almost never reproduce because they usually die before they reach reproductive age. Why, then, is the S allele not eliminated from the central African population by selection rather than being maintained at such high levels? As it turns out, one of the leading causes of illness and death in central Africa, especially among young children, is malaria. People who are heterozygous for the sickle cell allele (and thus do not suffer from sickle cell anemia) are much less susceptible to malaria. The reason is that when the parasite that causes malaria, Plasmodium falciparum, enters a red blood cell, it causes extremely low oxygen tension in the cell, which leads to sickling in cells of individuals either homozygous or heterozygous for the sickle cell allele (but not in individuals that do not have the sickle cell allele). Such cells are quickly filtered out of the bloodstream by the spleen, thus eliminating the parasite (the spleen’s filtering effect is what leads to anemia in persons homozygous for the sickle cell allele because large numbers of red blood cells become sickle-shaped and are removed; in the case of heterozygotes, only those cells containing the Plasmodium parasite sickle, whereas the remaining cells are not affected, and thus anemia does not occur). Thus, homozygotes with the sickle cell allele all die of anemia, homozygotes for the non-sickle cell allele are vulnerable to malaria, and heterozygotes have the highest fitness. Consequently, even though most homozygous recessive individuals die at a young age, the sickle cell allele is maintained at high levels in these populations because it is associated with resistance to malaria in heterozygotes and also, for reasons not yet fully understood, with increased fertility in female heterozygotes. Figure 20.17 shows the overlap between regions where sickle cell anemia is found and those where malaria is prevalent. For people living in areas where malaria is common, having the sickle cell allele in the heterozygous condition has adaptive value. However, in the United States, the environment does not place a premium on resistance to malaria because the disease is now essentially absent from North America. Consequently, no adaptive value counterbalances the ill effects of the disease; in this nonmalarial environment, selection is acting to eliminate the S allele. Only 1 in 375 Americans of African descent, many of whose ancestors lived in central Africa, develops sickle cell anemia, far fewer than in central Africa.

Learning Outcomes Review 20.6

Selection can maintain variation within populations in a number of ways. Negative frequency-dependent selection tends to favor rare phenotypes. Oscillating selection favors different phenotypes at different times. In some cases, heterozygotes have a selective advantage that may act to retain alleles that are deleterious in the homozygous state. ■■ ■■

How would genetic variation in a population change if heterozygotes had the lowest fitness? Explain the difference between negative frequencydependent and oscillating selection. Over long periods of time, which is more likely to maintain variation in a population?

432 part IV  Evolution

20.7 Selection

Acting on Traits Affected by Multiple Genes

Learning Outcomes 1. Define and contrast disruptive, directional, and stabilizing selection. 2. Explain the evolutionary outcome of each of these types of selection.

In nature, many traits—perhaps most—are affected by more than one gene. The interactions between genes are typically complex, as you saw in chapter 12. For example, alleles of hundreds of genes play a role in determining human height (see figure 12.10). In such cases, selection operates on all the genes, influencing most strongly those that make the greatest contribution to the phenotype. Such traits exhibit a continuous distribution of phenotypes, often conforming to a normal (“bell”) curve, rather than to several discretely different phenotypes (such as “red” and “black”).

Disruptive selection removes intermediates In some situations, selection acts to eliminate intermediate types, a phenomenon called disruptive selection (figure 20.18a). A clear example is the different beak sizes of the ­African black-­bellied seedcracker finch (figure 20.19). Populations of these birds contain individuals with large and small beaks, but very few individuals with intermediate-sized beaks. As their name implies, these birds feed on seeds, and the available seeds fall into two size categories: large and small. Only large-beaked birds can open the tough shells of large seeds, whereas birds with the smaller beaks are more adept at handling small seeds. Birds with intermediate-sized beaks are at a disadvantage with both seed types—they are unable to open large seeds and too clumsy to efficiently process small seeds. Consequently, selection acts to eliminate the intermediate phenotypes, in effect partitioning (or “disrupting”) the population into two phenotypically distinct groups.

Directional selection eliminates phenotypes at one end of a range When selection acts to eliminate one extreme from an array of phenotypes, the genes promoting this extreme become less frequent in the population and may eventually disappear. This form of selection is called directional selection (see ­f igure  20.18b). Thus, in the Drosophila population illustrated in f­ igure 20.20, eliminating flies that move toward light causes the population over time to contain fewer individuals with alleles promoting such behavior. If you were to pick an individual at random from a later generation of flies, there is a smaller chance that the fly would spontaneously move toward light than if you had selected a fly from the original population. Artificial selection has changed the population in the direction of being

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Figure 20.18 Three kinds of selection. The top panels show the populations before selection has occurred (under the solid red line).

Within the population, those favored by selection are shown in light brown. The bottom panels indicate what the populations would look like in the next generation. The dashed red lines are the distribution of the original population and the solid, dark brown lines are the true distribution of the population in the next generation. a. In disruptive selection, individuals in the middle of the range of phenotypes of a certain trait are selected against, and the extreme forms of the trait are favored. b. In directional selection, individuals concentrated toward one extreme of the array of phenotypes are favored. c. In stabilizing selection, individuals with midrange phenotypes are favored, with selection acting against both ends of the range of phenotypes.

Question: Does disruptive selection promote differences in beak size

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in the African black-bellied seedcracker finches (Pyrenestes ostrinus)?

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SCIENTIFIC THINKING 11 10 9 8 7 6 5 4 3 2 1 0 Field Study: Capture, measure, and release birds in a population. Follow the birds through time to determine how long each lives. Result: Large- and small-beaked birds have higher survival rates than birds with intermediate-sized beaks. Interpretation: What would happen if the distribution of seed size and hardness in the environment changed?

Figure 20.19 Disruptive selection for large and small beaks. Differences in beak size in the black-bellied seedcracker

finch of west Africa are the result of disruptive selection.

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Figure 20.20 Directional selection for negative phototropism in Drosophila. Flies that moved toward light were

discarded, and only flies that moved away from light were used as parents for the next generation. This procedure was repeated for 20 generations, producing substantial evolutionary change.

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Inquiry question What would happen if after 20

generations, experimenters started keeping flies that moved toward the light and discarded the others?

chapter

20 Genes Within Populations 433

less attracted to light. Directional selection often occurs in nature when the environment changes, favoring traits at one end of the phenotypic distribution.

Stabilizing selection favors individuals with intermediate phenotypes When selection acts to eliminate both extremes from an array of phenotypes, the result is to increase the frequency of the already common intermediate type. This form of selection is called stabilizing selection (see figure 20.18c). In effect, selection is operating to prevent change away from this middle range of values. Selection does not change the most common phenotype of the population, but rather makes it even more common by eliminating extremes. Many examples are known. In humans, infants with intermediate weight at birth have the highest survival rate (figure 20.21). In ducks and chickens, eggs of intermediate weight have the highest hatching success.

Learning Outcomes Review 20.7

In disruptive selection, intermediate forms of a trait diminish; in stabilizing selection, intermediates increase, whereas in disruptive selection they decrease. Directional selection shifts frequencies toward one end or the other and may eventually eliminate alleles entirely. ■■ How does directional selection differ from frequencydependent selection?

20.8

Experimental Studies of Natural Selection

Learning Outcome 1. Explain how experiments can be used to test evolutionary hypotheses.

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lowest at an intermediate birth weight; both smaller and larger babies have a greater tendency to die than those around the most frequent weight (tan area; left y-axis) of between 7 and 8 pounds. Recent medical advances have reduced mortality rates for small and large babies.

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Inquiry question As improved medical technology leads to decreased infant mortality rates, how would you expect the distribution of birth weights in the population to change?

Data analysis Sketch what a graph would look like that showed the absolute number of infant mortality deaths versus birth weight. 434 part IV  Evolution

To study evolution, biologists have traditionally investigated what has happened in the past, sometimes many millions of years ago. To learn about dinosaurs, a paleontologist looks at dinosaur fossils. To study human evolution, an anthropologist looks at human fossils and, increasingly, examines the “family tree” of mutations that have accumulated in human DNA over millions of years. In this traditional approach, evolutionary biology is similar to astronomy and history, relying on observation rather than experimentation to examine ideas about past events. Nonetheless, evolutionary biology is not entirely an observational science. Darwin was right about many things, but one area in which he was mistaken concerns the pace at which evolution occurs. Darwin thought that evolution occurred at a very slow, almost imperceptible pace. But in recent years many case studies have demonstrated that in some circumstances, evolutionary change can occur rapidly. Consequently, experimental studies can be devised to test evolutionary hypotheses. Although laboratory studies on fruit flies and other organisms have been common for nearly a century, scientists have only recently started conducting experimental studies of evolution in nature. One excellent example of how observations of the natural world can be combined with rigorous experiments in the lab and in the field concerns research on guppies.

Guppy color variation in different environments suggests natural selection at work The guppy is a popular aquarium fish because of its bright coloration and prolific reproduction. In nature, guppies are found in small streams in northeastern South America and in many mountain streams on the nearby island of Trinidad. One interesting feature of several of the streams is that they have waterfalls. Amazingly, guppies and some other fish are capable of colonizing portions of the stream above the waterfall.

The killifish is a particularly­good colonizer; apparently on rainy nights, it will wriggle out of the stream and move through the damp leaf litter. Guppies are not so proficient, but they are good at swimming upstream. During flood seasons, rivers sometimes overflow their banks, creating secondary channels that move through the forest. On these occasions, guppies may be able to swim upstream in the secondary channels and invade the pools above waterfalls. By contrast, some species are not capable of such dispersal and thus are found only in streams below the first waterfall. One species whose distribution is restricted by waterfalls is the pike cichlid, a voracious predator that feeds on other fish, including guppies. Because of these barriers to dispersal, guppies can be found in two very different environments. In pools just below the waterfalls, predation by the pike cichlid is a substantial risk, and rates of survival are relatively low. But in similar pools just above the waterfall, the only predator present is the killifish, which rarely preys on guppies. Guppy populations above and below waterfalls exhibit many differences. In the ­high-predation pools, guppies exhibit drab coloration. Moreover, they tend to reproduce at a younger age and attain relatively smaller adult sizes. Male fish above the waterfall, in contrast, are colorful (figure 20.22), mature later, and grow to larger sizes. These differences suggest the operation of natural selection. In the low-predation environment, males display gaudy colors and spots that they use to court females. Moreover, larger males are most successful at holding territories and mating with females, and larger females lay more eggs. Thus, in the absence of predators, larger and more colorful fish may have produced more offspring, leading to the evolution of those traits. In pools below the waterfall, natural selection would favor different traits. Colorful males are likely to attract the attention of the pike cichlid, and high predation rates mean that most fish live short lives. Individuals that are more drab and shunt energy into early reproduction, rather than growth to a larger size, are therefore likely to be favored by natural selection.

Experimentation reveals the agent of selection Although the differences between guppies living above and below the waterfalls suggest evolutionary responses to differences in the strength of predation, alternative explanations are possible. Perhaps, for example, only very large fish are capable of crawling past the waterfall to colonize pools. If this were the case, then a founder effect would occur in which the new population was established solely by individuals with genes for large size. The only way to rule out such alternative possibilities is to conduct a controlled experiment.

The laboratory experiment The first experiments were conducted in large pools in laboratory greenhouses. At the start of the experiment, a group of 2000 guppies was divided equally among 10 large pools. Four weeks later, pike cichlids were added to four of the pools and killifish to another four, with the remaining two pools left to serve as “no-predation” controls. Fourteen months later (which corresponds to 10 guppy generations), the scientists compared the populations. The guppies in the killifish and control pools were indistinguishable—brightly colored and large. In contrast, the guppies in the pike cichlid pools were smaller and drab in coloration (figure 20.23).

Killifish (Rivulus hartii )

Pike cichlid (Crenicichla alta)

Guppy (Poecilia reticulata)

Guppy (Poecilia reticulata)

Figure 20.22 The evolution of protective coloration in guppies. In pools below waterfalls where predation is high, male

guppies are small and drab in color. In the absence of the highly predatory pike cichlid in pools above waterfalls, male guppies are larger and much more colorful and attractive to females. The killifish is also a predator, but it only rarely eats guppies. The evolution of these differences in guppies can be experimentally tested.

These results established that predation can lead to rapid­ evolutionary change, but do these laboratory experiments reflect what occurs in nature?

The field experiment To find out whether the laboratory results were an accurate reflection of natural processes, the scientists located two streams that had guppies in pools below a waterfa ll, but not above it. As in other Trinidadian streams, the pike cichlid was present in the lower pools, but only the killifish was found above the waterfalls. The scientists then transplanted guppies to the upper pools and returned at several-year intervals to monitor the populations. Despite originating from populations in which predation levels were high, the transplanted populations rapidly evolved the traits characteristic of low-predation guppies: they matured late, attained greater size, and had brighter colors. The control populations in the lower pools, by contrast, continued to be drab and to mature early and at a smaller size. Laboratory analysis confirmed that the variations between the populations were the result of genetic differences. These results demonstrate that substantial evolutionary change can occur in just a few years. More generally, these studies indicate how scientists can formulate hypotheses about how evolution occurs and then test these hypotheses in natural conditions. chapter

20 Genes Within Populations 435

SCIENTIFIC THINKING Question: Does the presence of predators affect the evolution of guppy color? Hypothesis: Predation on the most colorful individuals will cause a

20.9  Interactions

Among Evolutionary Forces

population to become increasingly dull through time. Conversely, in populations with few or no predators, increased color will evolve. Experiment: Establish laboratory populations of guppies in large pools with or without predators. Result: The populations with predators evolved to have fewer spots, while the populations in pools without predators evolved more spots.

no predation low predation high predation

14

Spots per Fish

12 11

Mutation and genetic drift may counter selection

10 9 8 0

4 8 Duration of Experiment (months)

12

Interpretation: Why does color increase in the absence of predators? How would you test your hypothesis?

Figure 20.23 Evolutionary change in spot number. 

Guppy populations raised for 10 generations in low-predation or no-predation environments in laboratory greenhouses evolved a greater number of spots, whereas selection in more dangerous environments, such as the pools with the highly predatory pike cichlid, led to less conspicuous fish. The same results are seen in field experiments conducted in pools above and below waterfalls.

Inquiry question How do these results depend

on the manner by which the guppy predators locate their prey?

The results give strong support to the theory of evolution by natural selection.

Learning Outcomes Review 20.8

Although much of evolutionary theory is derived from observation, experiments are sometimes possible in natural settings. Studies have revealed that traits can shift in populations in a relatively short time. The data obtained from evolutionary experiments can be used to refine theoretical assumptions. ■■

1. Discuss how evolutionary processes can work simultaneously, but in opposing ways. 2. Evaluate what determines the evolutionary outcome when multiple processes are operating simultaneously.

The amount of genetic variation in a population may be determined by the relative strength of different evolutionary processes. Sometimes these processes act together, and in other cases they work in opposition.

13

?

Learning Outcomes

What experiments could you design to test other examples of natural selection, such as the evolution of pesticide resistance or background color matching?

436 part IV  Evolution

In theory, if allele B mutated to allele b at a high enough rate, allele b could be maintained in the population, even if natural selection strongly favored allele B. In nature, however, mutation rates are rarely high enough to counter the effects of natural selection. The effect of natural selection also may be countered by genetic drift. Both of these processes may act to remove variation from a population. But selection is a nonrandom process that operates to increase the representation of alleles that enhance survival and reproductive success, whereas genetic drift is a random process in which any allele may increase. Thus, in some cases, drift may lead to a decrease in the frequency of an allele that is favored by selection. In some extreme cases, drift may even lead to the loss of a favored allele from a population. Remember, however, that the magnitude of drift is inversely related to population size; consequently, natural selection is expected­to overwhelm drift, except when populations are very small.

Gene flow may promote or constrain evolutionary change Gene flow can be either a constructive or a constraining force. On one hand, gene flow can spread a beneficial mutation that arises in one population to other populations. On the other hand, gene flow can impede adaptation within a population by the continual flow of inferior alleles from other populations. Consider two populations of a species that live in different environments. In this situation, natural selection might favor different alleles—B and b—in the two populations. In the absence of other evolutionary processes such as gene flow, the frequency of B would be expected to reach 100% in one population and 0% in the other. However, if gene flow occurred between the two populations, then the less favored allele would continually be reintroduced into each population. As a result, the frequency of the

Index of Copper Tolerance

alleles in the populations would reflect a balance between the rate at which gene flow brings the inferior allele into a population, and the rate at which natural selection removes it. A classic example of gene flow opposing natural selection occurs on abandoned mine sites in Great Britain. Although mining activities ceased hundreds of years ago, the concentration of metal ions in the soil is still much greater than in surrounding areas. Large concentrations of heavy metals are generally toxic to plants, but alleles of certain genes confer the ability to grow on soils high in heavy metals. The ability to tolerate heavy metals comes at a price, however; individuals with the resistance allele exhibit lower growth rates on nonpolluted soil. Consequently, we would expect the resistance allele to occur with a frequency of 100% on former mine sites and 0% elsewhere. Heavy-metal tolerance has been studied intensively in the slender bent grass, in which the resistance allele occurs at intermediate levels in many areas (figure 20.24). The explanation relates to the reproductive system of this grass, in which pollen, the floral equivalent of sperm, is dispersed by the wind. As a result, pollen grains—and the alleles they c­arry—can move great distances, leading to levels of gene flow between mine sites and unpolluted areas high enough to counteract the effects of natural selection. In general, the extent to which gene flow can hinder the effects of natural selection should depend on the relative strengths of the two processes. In species in which gene flow is generally strong, such as in birds and wind-pollinated plants, the frequency of the allele less favored by natural selection may be relatively high. In more sedentary species that exhibit low levels of gene flow, such as snails, the favored allele should occur at a frequency near 100%.

Pollen Prevailing wind 60

Nonmine

Mine site

Nonmine Bent grass (Agrostis tenuis)

40 20 0

20

0

0

20

40

60

Distance Upwind from Mine (m)

80 100 120 140 160

Distance Away from Mine (m)

Figure 20.24 Degree of copper tolerance in grass plants on and near ancient mine sites. Individuals with tolerant alleles

have decreased growth rates on unpolluted soil. Thus, we would expect copper tolerance to be 100% on mine sites and 0% on nonmine sites. However, prevailing winds blow pollen-containing nontolerant alleles onto the mine site and tolerant alleles beyond the site’s borders. The amount of pollen received decreases with distance, which explains the changes in levels of tolerance. The index of copper tolerance is calculated as the growth rate of a plant on soil with high concentrations of copper relative to growth rate on soils with low levels of copper; the higher the index, the more tolerant the plant is of heavy metal pollution.

Data analysis Examine the index of copper tolerance on nonmine areas. What does it suggest about the factor responsible? In particular, how does the index change as a function of distance from the mine on the right-hand side? And how does the relationship between distance and index value differ on the two sides of the mine? What process might be responsible for such patterns?

Learning Outcomes Review 20.9

Allele frequencies sometimes reflect a balance between opposing processes. Gene flow, for example, may increase some alleles while natural selection decreases them. Where several processes are involved, observed frequencies depend on the relative strength of the processes. ■■

Under what circumstances might evolutionary processes operate in the same direction, and what would be the outcome?

20.10 The

Limits of Selection

Learning Outcomes 1. Define pleiotropy and epistasis. 2. Explain how these phenomena may affect the evolutionary response to selective pressure.

Although selection is the most powerful of the principal agents of genetic change, there are limits to what it can accomplish. These

limits result from multiple phenotypic effects of alleles, lack of genetic variation upon which selection can act, and interactions between genes.

Genes have multiple effects Alleles often affect multiple aspects of a phenotype (the phenomenon of pleiotropy; see chapter 12). These multiple effects tend to set limits on how much a phenotype can be altered. For example, in chickens, the same gene affects the size of a hen’s comb and the rate at which she lays—alleles that produce large combs also lead to faster egg production. As a result of this linkage, selection for hens that lay many eggs, but have a small comb, would be very difficult.

Evolution requires genetic variation Genetic variation is a prerequisite for evolutionary change; without it, natural selection cannot produce evolution. Over 80% of the gene pool of the thoroughbred horses racing today goes back to 31 ancestors from the late 18th century. Despite intense directional selection on thoroughbreds, their performance chapter

20 Genes Within Populations 437

170

Finish time (seconds)

160

150

140

130

120

110

1880

1900

1920

1940

1960

1980

2000

2020

Figure 20.25  Selection for increased speed in racehorses is no longer effective. Kentucky Derby winning speeds have not improved significantly since 1950.

?

Inquiry question What might explain the lack of change in winning speeds?

times have not improved for more than 60 years (figure 20.25). Decades of intense selection presumably have removed variation from the population at a rate greater than mutation can replenish it, such that little genetic variation now remains, and evolutionary change is not possible. In some cases, phenotypic variation for a trait may never have had a genetic basis. The compound eyes of insects are made up of hundreds of visual units, termed ommatidia (described in chapter 33). In some individuals, the left eye contains more ommatidia than the right. In other individuals, the right eye contains more than the left (figure 20.26). However, despite intense selection experiments in the laboratory, scientists have never been able to produce a line of fruit flies that consistently has more ommatidia in the left eye than in the right. The reason is that separate genes do not exist for the left and right eyes. Rather, the same genes affect both eyes, and differences in the number of ommatidia result from differences that occur as the eyes are formed in the development process. Thus, despite the existence of phenotypic variation, no underlying genetic variation is available for selection to favor.

Gene interactions affect fitness of alleles As discussed in chapter 12, epistasis is the phenomenon in which an allele for one gene may have different effects, depending on alleles present at other genes. Because of epistasis, the selective 438 part IV  Evolution

Right eye of insect

Left eye of insect

Ommatidia

Figure 20.26 Phenotypic variation in insect ommatidia.  In some individuals, the number of ommatidia in the left eye is greater than the number in the right.

advantage of an allele at one gene may vary from one genotype to another. If a population is polymorphic for a second gene, then selection on the first gene may be constrained because different alleles are favored in different individuals of the same population. Studies on bacteria illustrate how selection on alleles for one gene can depend on which alleles are present at other genes. In E. coli, two biochemical pathways exist to break down gluconate, each using enzymes produced by different genes. One gene produces the enzyme 6-PGD, for which there are several alleles.

When the common allele for the second gene, which codes for the other biochemical pathway, is present, selection does not favor one allele over another at the 6-PGD gene. In some E. coli, however, an alternative allele at the second gene occurs that is not functional. The bacteria with this alternative allele are forced to rely only on the 6-PGD pathway, and in this case, selection favors one 6-PGD allele over another. Thus, epistatic interactions exist between the two genes, and the outcome of natural selection on the 6-PGD gene depends on which alleles are present at the second gene.

Learning Outcomes Review 20.10

In pleiotropy, a single gene affects multiple traits; in epistasis, interaction between alleles of different genes affects a single trait. Both these conditions can constrain the effects of natural selection. ■■

How can epistasis and pleiotropy constrain the evolutionary response to natural selection?

Chapter Review tamoncity/Shutterstock

20.1 Genetic Variation and Evolution Many processes can lead to evolutionary change. Darwin proposed that evolution of species occurs by the process of natural selection. Other processes can also lead to evolutionary change. Populations contain ample genetic variation. For a population to be able to evolve, it must contain genetic variation. DNA testing shows that natural populations generally have substantial variation.

20.2 Changes in Allele Frequency (figure 20.3) The Hardy–Weinberg principle allows prediction of genotype frequencies. Hardy–Weinberg equilibrium exists when observed genotype frequencies match the prediction from calculated frequencies. It occurs only when evolutionary processes are not acting to shift the distribution of alleles or genotypes in the population. Hardy–Weinberg predictions can be applied to data to find evidence of evolutionary processes. If genotype frequencies are not in Hardy–Weinberg equilibrium, then evolutionary processes must be at work.

20.3 Five Agents of Evolutionary Change (figure 20.4) Mutation changes alleles. Mutations are the ultimate source of genetic variation. Because mutation rates are low, mutation usually is not responsible for deviations from Hardy–Weinberg equilibrium. Gene flow occurs when alleles move between populations. Gene flow is the migration of new alleles into a population. It can introduce genetic variation and can homogenize allele frequencies between populations. Nonrandom mating shifts genotype frequencies. Assortative mating, in which similar individuals tend to mate, increases homozygosity; disassortative mating increases the frequency of heterozygotes.

Genetic drift may alter allele frequencies in small populations. Genetic drift refers to random shifts in allele frequency. Its effects may be severe in small populations. Selection favors some phenotypes over others. For evolution by natural selection to occur, genetic variation must exist, it must result in differential reproductive success, and it must be inheritable.

20.4 Quantifying Natural Selection A phenotype with greater fitness usually increases in frequency. Fitness is defined as the reproductive success of an individual. Relative fitness refers to the success of one phenotype relative to others in a population. Usually, the phenotype with highest relative fitness increases in frequency in the next generation, assuming that phenotypic differences are the result of genetic differences. Fitness may consist of many components. Reproductive success is determined by how long an individual survives, how often it mates, and how many offspring it has per reproductive event.

20.5 Reproductive Strategies The sexes often have different reproductive strategies. One sex may be choosier than the other, and which one often depends on the degree of parental investment. Sexual selection occurs through mate competition and mate choice. Intrasexual selection involves competition among members of the same sex for the chance to mate. Intersexual selection is one sex choosing a mate. Mate choice may provide direct benefits (increased resource availability or parental care) or indirect benefits (genetic quality of the mate).

20.6 Natural Selection’s Role in Maintaining Variation Frequency-dependent selection may favor either rare or common phenotypes. Negative frequency-dependent selection favors rare phenotypes and maintains variation within a population. Positive frequency-dependent selection favors the common phenotype and leads to decreased variation. chapter

20 Genes Within Populations 439

Experimentation reveals the agent of selection. Guppies in natural populations subject to different predators were shown to undergo color change over generations.

In oscillating selection, the favored phenotype changes as the environment changes. If environmental change is cyclical, selection would favor first one phenotype, then another, maintaining variation.

20.9 Interactions Among Evolutionary Forces

In some cases, heterozygotes may exhibit greater fitness than homozygotes. Heterozygote advantage favors individuals with both alleles.

Mutation and genetic drift may counter selection. In theory, a high rate of mutation could oppose natural selection, but this rarely happens. Genetic drift also can work counter to natural selection.

20.7 Selection Acting on Traits Affected by Multiple Genes

Gene flow may promote or constrain evolutionary change. Gene flow can spread a beneficial mutation to other populations, but it can also impede adaptation due to influx of alleles with low fitness in a population’s environment.

Disruptive selection removes intermediates. When intermediate phenotypes are at a disadvantage, a population may exhibit a bimodal trait distribution. Directional selection eliminates phenotypes at one end of a range. Directional selection tends to shift the mean value of the population toward the favored end of the distribution.

20.10 The Limits of Selection Genes have multiple effects. Pleiotropic genes, which have multiple effects, set limits on how much a phenotype can be altered. Even if one affected trait is favored, other affected traits may not be.

Stabilizing selection favors individuals with intermediate phenotypes. Stabilizing selection eliminates both extremes and increases the frequency of an intermediate type. The population may have the same mean value, but with decreased variation.

Evolution requires genetic variation. Intense selection pressure may remove genetic variation.

20.8 Experimental Studies of Natural Selection

Gene interactions affect fitness of alleles. In epistasis, fitness of one allele may vary depending on the genotype of a second gene.

The hypothesis that natural selection leads to evolutionary change can be tested experimentally. Guppy color variation in different environments suggests natural selection at work. In the presence of predators, males are much less colorful.

Visual Summary Single genes Genetic variation in populations

produces

Phenotypic variation

affected by Multiple genes

changes leading to Pleiotropy Epistasis

Deviations from HardyWeinberg equilibrium

Factors that limit evolution

Lack of genetic variation

indicating action of

constrained by

Processes that cause evolutionary change include

Mutation

Gene flow

Nonrandom mating

Genetic drift

Natural selection

occurs when phenotypes differ in Fitness differences result from

Survival

Mating success determined by Reproductive strategies

440 part IV  Evolution

Number of offspring per mating event

Review Questions tamoncity/Shutterstock

U N D E R S TA N D

A P P LY

1. Assortative mating a. b. c. d.

1. In a population of red (dominant allele) or white flowers in Hardy–Weinberg equilibrium, the frequency of red flowers is 91%. What is the frequency of the red allele?

affects genotype frequencies expected under Hardy– Weinberg equilibrium. affects allele frequencies expected under Hardy–Weinberg equilibrium. has no effect on the genotypic frequencies expected under Hardy–Weinberg equilibrium because it does not affect the relative proportion of alleles in a population. increases the frequency of heterozygous individuals above Hardy–Weinberg expectations.

a. 9% b. 30%

2. Genetic drift and natural selection can both lead to rapid rates of evolution. However, a. b. c.

2. When the environment changes from year to year and different phenotypes have different fitness in different environments, a. b. c. d.

d.

natural selection will operate in a frequency-dependent manner. the effect of natural selection may oscillate from year to year, favoring alternative phenotypes in different years. genetic variation is not required to get evolutionary change by natural selection. None of the choices is correct.

genetic drift works fastest in large populations. only drift leads to adaptation. natural selection requires genetic drift to produce new variation in populations. both processes of evolution can be slowed by gene flow.

3. Suppose that the relationship between birth weight and infant mortality, instead of being at a minimum at intermediate sizes, changed such that babies born at 5 or 10 pounds had the lowest mortality, with an increase in between such that 7.5-pound babies had higher mortality. How would you expect the distribution of birth weights to change over time?

3. Many factors can limit the ability of natural selection to cause evolutionary change, including a. b. c. d.

c. 91% d. 70%

a. b. c.

a conflict between traits favored by reproduction and survival. lack of genetic variation. pleiotropy. All of the choices are correct.

d.

It would not change. The distribution would shift to the right. The distribution would become bimodal, with two peaks and the mean value unchanged. The distribution would become bimodal, with two peaks and the mean value shifted to the right.

4. Stabilizing selection differs from directional selection because a.

in the former, phenotypic variation is reduced but the average phenotype stays the same, whereas in the latter both the variation and the mean phenotype change. b. the former requires genetic variation, but the latter does not. c. intermediate phenotypes are favored in directional selection. d. None of the choices is correct.

births in population infant mortality

a. expected only in large populations. b. mechanisms that increase genetic variation in a population. c. two different modes of natural selection. d. forms of genetic drift. 6. Relative fitness

b. c. d.

refers to the survival rate of one phenotype compared to that of another. is the physical condition of an individual’s siblings and cousins. refers to the reproductive success of a phenotype. None of the choices is correct.

15

50 30 20

10

10 7

5

Percent Infant Mortality

Percent of Births in Population

70

5. Founder effects and bottlenecks are

a.

100

20

5 3 2 2



3

4

5

6

7

8

9

10

Birth Weight in Pounds

7. For natural selection to result in evolutionary change, a. b. c. d.

variation must exist in a population. reproductive success of different phenotypes must differ. variation must be inherited from one generation to the next. All of the choices are correct.

8. The elaborate tail feathers of a male peacock evolved because they a. b. c. d.

improve reproductive success of males and females. improve male survival. reduce survival. None of the choices is correct.

4. Peahens prefer to mate with peacocks that have more eyespots in their tail feathers (that is, longer tail feathers). It has also been suggested that the longer the tail feathers, the more impaired the flight of the males. One possible hypothesis to explain such a preference by females is that the males with the longest tail feathers experience the most severe handicap, and if they can nevertheless survive, it reflects their “vigor.” Suggest some studies that would allow you to test this idea. Your description should include the kinds of traits that you would measure and why. (Refer to figure 20.10.) chapter

20 Genes Within Populations 441

SYNTHESIZE 1. In Trinidadian guppies a combination of elegant laboratory and field experiments builds a very compelling case for predatorinduced evolutionary changes in color and life history traits. It is still possible, although not likely, that there are other differences between the sites above and below the falls aside from whether predators are present. What additional studies could strengthen the interpretation of the results? 2. On large black lava flows in the deserts of the southwestern United States, populations of many types of animals are composed primarily of black individuals. By contrast, on small lava flows, populations often have a relatively high proportion of light-colored individuals. How can you explain this difference?

442 part IV  Evolution

3. Based on a consideration of how strong artificial selection has helped eliminate genetic variation for speed in thoroughbred horses, we are left with the question of why, for many traits like speed (continuous traits), there is usually abundant genetic variation. This is true even for traits we know are under strong selection. Where does genetic variation ultimately come from, and how does the rate of production compare with the strength of natural selection? What other mechanisms can maintain and increase genetic variation in natural populations?

21 CHAPTER

The Evidence for Evolution Chapter Contents 21.1

The Beaks of Darwin’s Finches: Evidence of Natural Selection

21.2

Peppered Moths and Industrial Melanism: More Evidence of Selection

21.3

Artificial Selection: Human-Initiated Change

21.4

Fossil Evidence of Evolution

21.5

Anatomical Evidence for Evolution

21.6

Convergent Evolution and the Biogeographical Record

21.7

Darwin’s Critics

Georgia Southern University Museum

Introduction

Visual Outline Evidence for evolution includes evidence from

Selection

Fossils

Convergent evolution

Anatomy

categorized as Artificial Natural selection selection

Human

Cat

Porpoise

Jason Edwards/National Geographic/Getty Images

Bat

Horse

Biogeographical record

As we discussed in chapter 1, when Darwin proposed his revolutionary theory of evolution by natural selection, little actual evidence existed to bolster his case. Instead, Darwin relied on observations of the natural world, logic, and results obtained by breeders working with domestic animals. Since his day, however, the evidence for Darwin’s theory has become overwhelming. The case is built upon two pillars: first, evidence that natural selection can produce evolutionary change, and second, evidence from the fossil record that evolution has occurred. The whale skeleton pictured here is the Vogtle whale (Georgiacetus vogtlensis), which is the oldest whale fossil found in North America (40 million years old). The pelvic bones and hindlimbs reveal a link between land mammals and whales. In addition to evidence from studies of natural selection and fossils, information from many different areas of biology—fields as different as anatomy, molecular biology, and biogeography—is only interpretable scientifically as being the outcome of evolution.

21.1 The

Beaks of Darwin’s Finches: Evidence of Natural Selection

Learning Outcomes 1. Describe how the species of Darwin’s finches have adapted to feed in different ways. 2. Explain how climatic variation drives evolutionary change in the medium ground finch.

As you learned in chapter 20, a variety of processes can produce evolutionary change. Most evolutionary biologists, however, agree with Darwin’s thinking that natural selection is the primary process responsible for evolution. Although we cannot travel back through time, modern-day evidence allows us to test hypotheses about how evolution proceeds and confirms the power of natural selection as an agent of evolutionary change. This evidence comes from both the field and the laboratory and from both natural and human-altered situations. Darwin’s finches are a classic example of evolution by natural selection. When he visited the Galápagos Islands off the coast of Ecuador in 1835, Darwin collected 31 specimens of finches from three islands. Not being an expert on birds, Darwin had trouble identifying the specimens, believing by examining their beaks

Woodpecker finch (Cactospiza pallida)

that his collection contained wrens, “gross-beaks,” and blackbirds, all birds with which he was familiar from his native England. Upon Darwin’s return home, ornithologist John Gould informed Darwin that the species he had collected were not closely related to different English birds, but rather were a closely related group of distinct species, all similar to one another except for their beaks. In all, 14 species are now recognized.

Galápagos finches exhibit variation related to food gathering The diversity of Darwin’s finches is illustrated in figure 21.1. The ground finches feed on seeds that they crush in their powerful beaks; species with smaller and narrower beaks, such as the warbler finch, eat insects. Other species include fruit and bud eaters, and species that feed on cactus fruits and the insects they attract; some populations of the sharp-beaked ground finch even include “vampires” that sometimes creep up on seabirds and use their sharp beaks to pierce the seabirds’ skin and drink their blood. Perhaps most remarkable are the tool users, woodpecker finches that pick up a twig, cactus spine, or leaf stalk, trim it into shape with their beaks, and then poke it into dead branches to pry out grubs. The correspondence between the beaks of the finch species and their food sources suggested to Darwin that natural selection had shaped them. In The Voyage of the Beagle, written several years after his return to England, Darwin wrote, “Seeing this gradation and diversity of structure in one small, intimately related group of birds, one might really fancy that from an original paucity

Large ground finch (Geospiza magnirostris)

Cactus finch (Geospiza scandens)

Figure 21.1 Darwin’s finches. These species show differences in beaks and feeding habits among Darwin’s finches. This diversity arose when an ancestral finch colonized the islands and diversified into habitats lacking other types of small birds. The beaks of several species resemble those of different families of birds on the mainland. For example, the warbler finch has a beak very similar to that of warblers, to which it is not closely related.

Warbler finch (Certhidea olivacea)

444 part IV Evolution

Vegetarian tree finch (Platyspiza crassirostris)

of birds in this archipelago, one species has been taken and modified for different ends.”

Modern research has verified Darwin’s selection hypothesis Darwin’s observations suggest that differences among species in beak size and shape have evolved as the species adapted to use different food resources, but can this hypothesis be tested? In chapter 20, you read that the theory of evolution by natural selection requires that three conditions be met: 1. Phenotypic variation must exist in the population. 2. This variation must lead to differences among individuals in lifetime reproductive success. 3. Phenotypic variation among individuals must be genetically transmissible to the next generation. The key to successfully testing Darwin’s proposal proved to be patience. For more than 40 years, starting in 1973, Peter and Rosemary Grant of Princeton University and their students studied the medium ground finch on a tiny island in the center of the Galápagos called Daphne Major. These finches feed preferentially on small, tender seeds, produced in abundance by plants in wet years. During long periods of dry weather, the birds resort to larger, drier seeds, which are harder to crush. The Grants quantified beak shape among the medium ground finches of Daphne Major by carefully measuring beak depth (height of beak, from top to bottom, at its base) on individual birds.

G. fortis

11

Beak depth (mm)

10.0

9.5

9.0

Drought, large seeds become abundant

8.5

8.0 1970

a.

Wet years, small seeds become abundant

Beak depth of offspring (mm)

10.5

Measuring many birds every year, they were able to assemble for the first time a detailed portrait of evolution in action. The Grants found that not only did a great deal of variation in beak depth exist among members of the population, but the average beak depth changed from one year to the next in a predictable fashion. During droughts, plants produced few seeds, and all available small seeds were quickly eaten, leaving large seeds as the major remaining source of food. As a result, birds with shorter and deeper, more powerful beaks survived better, because they were better able to break open these large seeds (figure 21.2a). Conversely, in particularly wet years, plants flourished, producing an abundance of small seeds; as a result, birds with long and shallow beaks were favored, and beaks became pointier. Could these changes in beak dimension be evidence of evolution by natural selection? If so, the variation among individuals in beak size must be genetically based. An alternative possibility might be that the changes in beak depth do not reflect changes in gene frequencies, but rather are simply a response to diet—for example, perhaps crushing large seeds causes a growing bird to develop a larger beak. To rule out this possibility, the Grants tested for a genetic basis to beak size variation by measuring the relationship of parent beak size to offspring beak size, examining many broods over several years. The depth of the beak was very similar between parents and offspring regardless of environmental conditions (figure 21.2b), suggesting that the differences among individuals in beak size reflect genetic differences. This result satisfies condition number three mentioned above and therefore indicates that the year-to-year changes in average beak depth represent evolutionary change resulting from natural selection.

• •• • ••

10

•

9

8 8

1980

1990

••

• • •• • • • •

9

10

11

Mean beak depth of parents (mm)

b.

Figure 21.2 Evidence that natural selection alters beak shape in the medium ground finch, Geospiza fortis. a. In dry years, when only large, tough seeds are available, the mean beak depth increases. In wet years, when many small seeds are available, mean beak depth decreases. b. Beak depth is inherited from parents to offspring. Like many quantitative traits (see section 12.6), beak depth is probably determined by many genes and, on average, offspring tend to have a beak depth equal to the mean of their parents’ beak depth.

?

Inquiry question What would the relationship in figure 21.2b look like if differences in beak shape were affected not by genetic differences, but rather by an environmental factor, such as what a nestling bird ate during its growth period?

Data analysis Suppose that a male with a beak depth of 10 mm mated with a female with a beak depth of 8 mm. What would the expected beak depth of the offspring be? Would it matter if the female’s beak was 10 mm and the male’s 6 mm? chapter

21 The Evidence for Evolution 445

Learning Outcomes Review 21.1

Among Darwin’s finches, natural selection has been responsible for changes in the shape of the beak corresponding to characteristics of the available food supply. Because beak morphology is a genetically transmissible trait, a beak better suited to the distribution of available seed types would become more common in subsequent generations. ■■

Suppose that the act of eating hard seeds caused birds to develop bigger beaks. Would this lead to an evolutionary increase in beak size after a drought?

21.2 Peppered

Moths and Industrial Melanism: More Evidence of Selection

Learning Outcomes 1. Explain the relationship between pollution and color evolution in peppered moths. 2. Distinguish between demonstrating that evolution has occurred and understanding the mechanism that caused it.

When the environment changes, natural selection often may favor a trait that previously wasn’t favored. One classic example concerns the peppered moth, Biston betularia. Adults come in a range of shades, from light gray with black speckling (hence the name “peppered” moth) to jet black (melanic). Extensive genetic analysis has shown that the moth’s body color is a genetic trait that reflects different alleles of a single gene. Recent molecular genetic studies have demonstrated that

Figure 21.3 Tutt’s hypothesis explaining industrial melanism. 

These photographs show preserved specimens of the peppered moth, Biston betularia, placed on trees. Tutt proposed that the dark melanic variant of the moth is more visible to predators on unpolluted trees (left), whereas the light “peppered” moth is more visible to predators on bark blackened by industrial pollution (right). (Left) IanRedding/Shutterstock; (right) The Natural History Museum/Alamy Stock Photo

446 part IV Evolution

all-black individuals are the descendants of a single mutation. This dominant allele was present but very rare in populations before 1850. From that time on, dark individuals increased in frequency in moth populations near industrialized centers until they made up almost 100% of these populations. Biologists also noticed that in industrialized regions where the dark moths were common, the tree trunks were darkened almost black by the soot of pollution, which also killed many of the light-colored lichens on tree trunks.

Light-colored moths decreased in polluted areas Why did dark moths gain a survival advantage around 1850? In 1896, a British amateur moth collector named J. W. Tutt proposed what became the most commonly accepted hypothesis explaining the decline of the light-colored moths. He suggested that peppered forms were more visible to predators on sooty trees that have lost their lichens. Consequently, birds ate the peppered moths resting on the trunks of trees during the day. The black forms, in contrast, had an advantage because they were camouflaged (figure 21.3). Although Tutt initially had no evidence, British ecologist Bernard Kettlewell tested the hypothesis in the 1950s by releasing equal numbers of dark and light individuals into two sets of woods: one near heavily polluted Birmingham, and the other in unpolluted Dorset. If Tutt was correct, then dark moths should have survived better in the Birmingham woods and light moths in the Dorset woods. Kettlewell then set up lights in the woods to attract moths to traps to see how many of both kinds of moths survived. To identify the moths he had released, he had marked the released moths with a dot of paint on the underside of their wings, where birds could not see it. In the polluted area near Birmingham, Kettlewell recaptured only 19% of the light moths, but 40% of the dark ones. This indicated that dark moths survived better in these polluted woods, where tree trunks were dark. In the relatively unpolluted Dorset woods, Kettlewell recovered 12.5% of the light moths but only 6% of the dark ones. This result indicated that where the tree trunks were still light-­ colored, light moths’ survival was twice as great as that of dark moths.

When environmental conditions reverse, so does selection pressure In industrialized areas throughout Eurasia and North America, dozens of other species of moths have evolved in the same way as the peppered moth. The term industrial melanism refers to the phenomenon in which darker individuals come to predominate over lighter ones. In Great Britain, the air pollution that promoted industrial melanism began to reverse following enactment of the Clean Air Act in 1956. Beginning in 1959, the Biston population at Caldy Common outside Liverpool has been sampled each year. The frequency of the melanic (dark) form has dropped from a high of 93% in 1959 to less than 5% in 2002 (figure 21.4). The drop is consistent with a 15% selective disadvantage acting against moths with the dominant melanic allele; it correlates well with a significant drop in air pollution, particularly with a lowering of the levels of sulfur dioxide and suspended particulates, both of which act to darken trees. Interestingly, the same reversal of melanism occurred in the United States. Of 576 peppered moths collected at a field station near Detroit from 1959 to 1961, 515 were melanic, a frequency of 89%. The American Clean Air Act, passed in 1963, led to significant reductions in air pollution. Resampled in 2001, the frequency of melanics at the Detroit field station also had dropped to less than 5%; a similar decreasing trend was also observed in Pennsylvania (figure 21.4). The moth populations in Liverpool, Detroit, and Pensylvania, all part of the same natural experiment, exhibit strong evidence for natural selection.

The agent of selection may be difficult to pin down Although the evidence for natural selection in the case of the peppered moth is strong, Tutt’s hypothesis that predation on color-mismatched moths is the underlying mechanism of selection is currently being reevaluated. Researchers have noted­that the recent selection against melanism does not appear to correlate with changes in the abundance of light-colored tree lichens. At Caldy Common, the light form of the peppered moth began to increase in frequency long before lichens began to reappear on the trees. At the Detroit field station, the lichens never changed significantly as the dark moths first became dominant and then declined over a 30-year period. In fact, investigators have not been able to find peppered moths on Detroit trees at all, whether covered with lichens or not. Some evidence suggests the moths rest on leaves in the treetops during the day, but no one is sure. Could poisoning by pollution rather than predation by birds be the agent of natural selection on the moths? Perhaps—but to date, only selection resulting from bird predation has been demonstrated.

100 Percentage of melanic moths

Kettlewell later solidified his argument by placing moths on trees and filming birds looking for food. Sometimes the birds actually passed right over a moth that was the same color as its background. Recently, an enormous six-year study involving the release of nearly 5000 moths confirmed Kettlewell’s findings. Conducted in an unpolluted forest, the study found that dark-colored moths disappeared at a rate 10% higher than that of light-­colored moths. In addition, direct observations of 250 feeding events revealed that dark moths were captured by birds substantially more often than light-colored moths.

90 80 70 60 50 40 30 20 10 0 ‘59 ‘63 ‘67

‘71

‘75 ‘79 ‘83 ‘87 Year

‘91

‘95 ‘99 ‘03

Figure 21.4 Selection against melanism. The red circles indicate the frequency of melanic Biston betularia moths at Caldy Common in Great Britain. Yellow diamonds indicate frequencies of melanic B. betularia in Michigan, and the blue squares indicate corresponding frequencies in Pennsylvania.

?

Inquiry question What can you conclude from the fact that the frequency of melanic moths decreased to the same degree in the three locations?

Researchers supporting the bird predation hypothesis point out that a bird’s ability to detect moths may depend less on the presence or absence of lichens, and more on other ways in which the environment is darkened by industrial pollution. Pollution tends to cover all objects in the environment with a fine layer of particulate dust, which tends to decrease how much light is reflected by the surface. In addition, pollution has a particularly severe effect on birch trees, which are light in color. Both effects would tend to make the environment appear darker, and thus would favor darker moths by protecting them from predation by birds. Despite this uncertainty over the agent of selection, the overall pattern is clear. Kettlewell’s experiments established indisputably that selection favors dark moths in polluted habitats and light moths in pristine areas. The increase and subsequent decrease in the frequency of melanic moths, correlated with levels of pollution independently on two continents, demonstrates clearly that this selection drives evolutionary change. The current reconsideration of the agent of natural selection illustrates well the way in which scientific progress is achieved: hypotheses, such as Tutt’s, are put forth and then tested. If they are rejected, new hypotheses are formulated, and the process begins anew.

Learning Outcomes Review 21.2

Natural selection has favored the dark form of the peppered moth in areas subject to severe air pollution, perhaps because on darkened trees they are less easily seen by moth-eating birds. As pollution has abated, selection has in turn shifted to favor the light form. Although selection is clearly occurring, further research is required to understand whether predation by birds is the agent of selection. ■■

How would you test the idea that predation by birds is the agent of selection on moth coloration?

chapter

21 The Evidence for Evolution 447

21.3 Artificial

Selection: Human-Initiated Change

SCIENTIFIC THINKING Question: Can artificial selection lead to substantial evolutionary change? Hypothesis: Strong directional selection will quickly lead to a large shift in the mean value of the population. Experiment: In one population, every generation pick out the 20% of the population with the most bristles and allow them to reproduce to 20% with the smallest number of bristles.

1. Contrast the processes of artificial and natural selection. 2. Explain what artificial selection demonstrates about the power of natural selection.

Experimental selection produces changes in populations With the rise of genetics as a field of science in the 1920s and 1930s, researchers began conducting experiments to test the hypothesis that selection can produce evolutionary change. A favorite subject was the laboratory fruit fly, Drosophila melanogaster. Geneticists have imposed selection on just about every conceivable aspect of the fruit fly—including body size, eye color, growth rate, life span, and exploratory behavior—with a consistent result: selection for a trait leads to a strong and predictable evolutionary response. In one classic experiment, scientists selected for fruit flies with many bristles (stiff, hairlike structures) on their abdomens. At the start of the experiment, the average number of bristles was 9.5. Each generation, scientists picked out the 20% of the population with the greatest number of bristles and allowed them to reproduce, thus establishing the next generation. After 86 generations of this directional selection, the average number of bristles had quadrupled, to nearly 40! In another experiment, fruit flies in one population were selected for high numbers of bristles, while fruit flies in the other cage were selected for low numbers of bristles. Within 35 generations, the populations did not overlap at all in range of variation (figure 21.5). Similar experiments have been conducted on a wide variety of other laboratory organisms. For example, by selecting for rats that were resistant to tooth decay, in less than 20 generations scientists were able to increase the average time for onset of decay from barely over 100 days to greater than 500 days. 448 part IV Evolution

Low population

Number of Individuals 0

10

20

Mean

High population

Mean

Humans have imposed selection upon plants and animals since the dawn of civilization. Just as in natural selection, such a­ rtificial selection operates by favoring individuals with certain phenotypic traits, allowing them to reproduce and pass their genes on to the next generation. Assuming that phenotypic differences are genetically determined, this directional selection should lead to evolutionary change, and indeed it has. Artificial selection, imposed in laboratory experiments, agriculture, and the domestication process, has produced substantial change in almost every case in which it has been applied. This success is strong proof that selection is an effective evolutionary process.

Initial population

Mean

Learning Outcomes

form the next generation. In the other population, do the same with the

30 40 50 60 70 80 Bristle number in Drosophila

90 100

110

Result: After 35 generations, mean number of bristles has changed substantially in both populations. Interpretation: Note that at the end of the experiment, the range of variation lies outside the range seen in the initial population. Selection can move a population beyond its original range because mutation and recombination continuously introduce new variation into populations.

Figure 21.5 Artificial selection can lead to rapid and substantial evolutionary change.

?

Inquiry question What would happen if, within a

population, both small and large individuals were allowed to breed, but middle-sized ones were not?

Agricultural selection has led to extensive modification of crops and livestock Familiar livestock, such as cattle and pigs, and crops, such as corn and strawberries, are greatly different from their wild ancestors (figure 21.6). These differences have resulted from generations of human selection for desirable traits, such as greater milk production and larger corn ear size. An experiment with corn demonstrates the ability of artificial selection to rapidly produce major change in crop plants. In 1896, agricultural scientists began selecting for the oil content of corn kernels, which initially was 4.5%. Just as in the fruit fly experiments, the top 20% of all individuals were allowed to reproduce. By 1986, at which time 90 generations had passed, average oil content of the corn kernels had increased approximately 450%.

Figure 21.6 Corn looks very different from its ancestor. 

Teosinte

Intermediates

Modern corn

Teosinte, which can be found today in a remote part of Mexico, is very similar to the ancestor of modern corn. Artificial selection has transformed it into the form we know today.

Domesticated breeds have arisen from artificial selection

Fox Chihuahua

Wolf

Dachshund

Greyhound

Figure 21.7 Breeds of dogs. The differences among

dog breeds are greater­than the differences displayed among wild species of canids.

selectively breeding the tamest individuals, artificial selection has produced silver foxes that not only are as friendly as domestic dogs, but also exhibit many physical traits seen in dog breeds. Artyom

Geodakyan/ITAR-TASS News Agency/Alamy Stock Photo

Human-imposed selection has produced a great variety of breeds of cats, dogs (figure 21.7), pigeons, and other domestic animals. In some cases, breeds have been developed for particular purposes. Greyhound dogs, for example, resulted from s­ election for maximal running ability, resulting in an animal with long legs, a long tail for balance, an arched back to increase stride length, and great muscle mass. By contrast, the odd proportions of the ungainly dachshund resulted from selection for dogs that could enter narrow holes in pursuit of badgers. In other cases, varieties have been selected primarily for their appearance, such as the many colorful breeds of pigeons or cats. Domestication also has led to unintentional selection for some traits. In recent years, as part of an attempt to domesticate the silver fox, Russian scientists chose the most docile animals in each generation and allowed them to reproduce. Within 40 years, most foxes were exceptionally tame, not only allowing themselves to be petted, but also whimpering to get attention and sniffing and

Coyote

Figure 21.8 Domesticated foxes. After 40 years of

Mastiff

licking their caretakers (­figure 21.8). In many respects, they had become no different from domestic dogs. It was not only their behavior that changed, however. These foxes also began to exhibit other traits seen in some dog breeds, such as different color patterns, floppy ears, curled tails, and shorter legs and tails. Presumably, the genes responsible for docile behavior either affect these traits as well or are closely­linked to the genes for these other traits (the phenomena of pleiotropy and linkage, which are discussed in chapters 12 and 13).

Can selection produce major evolutionary changes? Given that we can observe the results of selection operating over a relatively short time, most scientists think that natural selection is the process responsible for the evolutionary changes documented in the fossil record. Some critics of evolution accept that selection can lead to changes within a species, but contend that such changes are relatively minor in scope and not equivalent to the substantial changes documented in the fossil record. In other words, it is one thing to change the number of bristles on a fruit fly or the size of an ear of corn, and quite another to produce an entirely new species. This argument does not fully appreciate the extent of change produced by artificial selection. Consider, for example, the existing breeds of dogs, all of which have been produced in the last few thousand years after wolves were domesticated, perhaps 15,000 years ago. If the various dog breeds did not exist and a paleontologist found fossils of animals similar to dachshunds, greyhounds, mastiffs, and chihuahuas, there is no question that they would be considered different species. Indeed, the differences in size and shape exhibited by these breeds are greater than those between members of different genera in the family Canidae—such as coyotes, jackals, foxes, and wolves—which have been evolving separately for 5 to 10 million years. Consequently,­the claim that artificial selection produces only minor changes is clearly incorrect. If selection operating over a ­ period of only a few thousand years can produce such substantial d­ifferences, it should be powerful enough, over the course of many millions of years, to produce the diversity of life we see around us today. chapter

21 The Evidence for Evolution 449

radioactive decay

Learning Outcomes Review 21.3

■■

In what circumstances might artificial selection fail to produce a desired change?

1 Proportion of parent isotope remaining

In artificial selection, humans choose which plants or animals to mate in an attempt to conserve desirable traits. Rapid and substantial results can be obtained over a very short time, often in a few generations. From this we can see that natural selection is capable of producing major evolutionary change.

0.75

Evidence of Evolution

Learning Outcomes 1. Describe how fossils are formed. 2. Explain the importance of the discovery of transitional fossils. 3. Name the evolutionary trends revealed by the study of horse evolution.

The most direct evidence that evolution has occurred is found in the fossil record. Today we have a far more complete understanding of this record than was available in Darwin’s time. Fossils are the preserved remains of once-living organisms. They include specimens preserved in amber, Siberian permafrost, and dry caves, as well as the more common fossils preserved as rocks. Rock fossils are created when three events occur. First, the organism must become buried in sediment; then, the calcium in bone or other hard tissue must mineralize; and finally, the surrounding sediment must eventually harden to form rock. The process of fossilization occurs only rarely. Usually, animal or plant remains decay or are scavenged before the process can begin. In addition, many fossils occur in rocks that are inaccessible to scientists. When they do become available, they are often destroyed by erosion and other natural processes before they can be collected. As a result, only a very small fraction of the species that have ever existed (estimated by some to be as many as 500 million) are known from fossils. Nonetheless, the fossils that have been discovered are sufficient to provide detailed information on the course of evolution through time.

The age of fossils can be estimated By dating the rocks in which fossils occur, we can get an accurate idea of how old the fossils are. In Darwin’s day, rocks were dated by their position with respect to one another (relative dating); rocks in lower strata are generally older because young rocks form on top of older ones. Knowing the relative positions of sedimentary rocks and the rates of erosion of different kinds of sedimentary rocks in different environments, geologists of the 19th century derived a fairly accurate idea of the relative ages of rocks. Today, geologists can determine the absolute age of rocks using isotopic dating. At the time a rock forms, some elements exist as different isotopes. Over time the less stable isotope is converted into the other isotope and the ratio of the two forms changes. 450 part IV Evolution

daughter isotope

Amount of daughter isotope 1 2

0.50

1 4

0.25 0

21.4 Fossil

parent isotope

Amount of parent isotope 0

1

1 8

2 3 Time in half-lives

1 16 4

5

Figure 21.9 Isotopic decay. Isotopes decay at a known rate, called their half-life. After one half-life, one-half of the original amount of parent isotope has transformed into a daughter isotope. After each successive half-life, one-half of the remaining amount of the parent isotope is transformed. For example, potassium is one of the most common atoms in organisms. All potassium (K) atoms have the same number of protons, but the isotopes of K vary in the number of neutrons they have. 40K has 19 protons and 21 neutrons and is less stable than 39K or 41K. 40K is converted (decays) over time and forms 40Ar (argon). The 40K half-life is 1.25 billion years. That is, it takes 1.25 billion years for the amount of 40K to decrease by 50%. The long half-life makes it useful for dating ancient fossils by determining the ratio of 40K to 40Ar (figure 21.9). For events that occurred more recently, radiocarbon dating can be used. Carbon in the form of atmospheric CO2, with a mix of isotopes 14C and 12C, is incorporated into plants via photosynthesis. The relative amount of 14C to 12C decreases with a half-life of about 5700 years. Other isotopes can be used for more intermediate dates.

Fossils present a history of evolutionary change When fossils are arrayed according to their age, from oldest to youngest, they often provide evidence of successive evolutionary change. At the largest scale, the fossil record documents the course of life through time, from the origin of prokaryotic and then eukaryotic organisms, through the evolution of fishes, the rise of land-dwelling organisms, the reign of the dinosaurs, and on to the origin of humans. In addition, the fossil record shows the waxing and waning of biological diversity through time, such as the periodic mass extinctions that have reduced the number of living species. These topics are discussed at greater length in chapter 25.

Fossils document evolutionary transitions Given the low likelihood of fossil preservation and recovery, it is not surprising that there are gaps in the fossil record. Nonetheless, intermediate forms are often available to illustrate how the major transitions in life occurred. Undoubtedly the most famous of these is the oldest known bird, Archaeopteryx (meaning “ancient feather”), which lived around 165 million years ago (mya) (figure 21.10). This species is

200 mya, oysters underwent a change from having small, curved shells to having larger, flatter ones, with progressively flatter fossils seen in the fossil record over a period of 12 million years. A host of other examples illustrate similar records of successive change. The demonstration of this successive change is one of the strongest lines of evidence that evolution has occurred.

The evolution of horses is a prime example of evidence from fossils

Figure 21.10 Fossil of Archaeopteryx, the first bird. 

The remarkable preservation of this specimen reveals soft parts usually not preserved in fossils; the presence of feathers like those of modernday birds, as well as other features, make clear that Archaeopteryx was a bird, despite the presence of many dinosaurian traits. To see a picture of what this animal may have looked like in life, see figure 34.29. Fossil evidence documenting the evolutionary descent of birds from dinosaurs is considered in greater detail in chapter 23 (see figure 23.12).

One of the most studied cases in the fossil record concerns the evolution of horses. Modern-day members of the family Equidae include horses, zebras, donkeys, and asses, all of which are large, long-legged, fast-running animals adapted to living on open grasslands. These species, all classified in the genus Equus, are the last living descendants of a long lineage that has produced 34 genera since its origin in the Eocene period, approximately 55 mya. Examination of these fossils has provided a particularly

Jason Edwards/National Geographic/Getty Images

clearly intermediate between birds and dinosaurs. Its feathers, similar in many respects to those of birds today, and other features ­clearly reveal that it is a bird. Nonetheless, in many other respects— for ­example, possession of teeth, a bony tail, and other anatomical characteristics—it is indistinguishable from some carnivorous ­ dinosaurs. Indeed, it is so similar to these dinosaurs that several specimens lacking preserved feathers were misidentified as ­dinosaurs and lay in the wrong natural history museum cabinet for ­several decades before the mistake was discovered! Archaeopteryx reveals a pattern commonly seen in intermediate fossils—rather than being intermediate in every trait, such fossils usually exhibit some traits like their ancestors and others like their descendants. In other words, traits evolve at different rates and different times; expecting an intermediate form to be intermediate in every trait would not be correct. The first Archaeopteryx fossil was discovered in 1859, the year Darwin published On the Origin of Species. Since then, paleontologists have continued to fill in the gaps in the fossil record. Today, the fossil record is far more complete, particularly among the vertebrates; fossils have been found linking all the major groups. Recent years have seen spectacular discoveries, closing some of the major remaining gaps in our understanding of vertebrate evolution. For example, four-legged aquatic mammals have been discovered that provide important insights concerning the evolution of whales and dolphins from land-dwelling, hoofed ancestors (figure 21.11). Similarly, fossil snakes with legs have shed light on the evolution of snakes, which are descended from lizards that gradually became more and more elongated with the simultaneous ­reduction and eventual disappearance of the limbs. In chapter 34, we discuss another recent discovery, Tiktaalik, a species that bridged the gap between fish and the first land-living vertebrates (see figure 34.17). On a finer scale, evolutionary change within some types of animals is known in exceptional detail. For example, about

Modern toothed whales

Rodhocetus kasrani's reduced hindlimbs could not have aided it in walking or swimming. Rodhocetus swam with an up-and-down motion, as do modern whales.

Ambulocetus natans probably walked on land (as do modern sea lions) and swam by flexing its backbone and paddling with its hindlimbs (as do modern otters).

Pakicetus attocki lived on land, but its skull differed from that of its ancestors and exhibited many characteristics seen in the skulls of whales today.

Figure 21.11 Whale “missing links.” The discoveries of

Ambulocetus, Rodhocetus, and Pakicetus have filled in the gaps between whales and their hoofed mammal ancestors. The features of Pakicetus illustrate that intermediate forms are not intermediate in all characteristics; rather, some traits evolve before others. In the case of the evolution of whales, changes occurred in the skull prior to evolutionary modification of the limbs. All three fossil forms occurred in the Eocene period, 45–55 mya. chapter

21 The Evidence for Evolution 451

Pleistocene

Miocene

Tooth size and shape The teeth of Hyracotherium were small and relatively simple in shape. Through time, horse teeth have increased greatly in length and have developed a complex pattern of ridges on their molars and premolars. The effect of these changes is to produce teeth better capable of chewing tough and gritty vegetation, such as grass, which tends to wear teeth down. As with body size, evolutionary change has not been constant through time. Rather, much of the change in tooth shape has occurred within the past 20 million years, and changes have not been constant across all horse lineages. All of these changes may be understood as adaptations to changing global climates. In particular, during the late Miocene and early Oligocene epochs (approximately 20 to 25 mya), grasslands became widespread in North America, where much of horse evolution occurred. As horses shifted from forests to grasslands, high-speed locomotion probably became more important to escape predators. By contrast, the greater flexibility provided by multiple toes and shorter limbs, which was advantageous for ducking through complex forest vegetation, was no longer beneficial. At 452 part IV Evolution

30 MYA

40 MYA Eocene

45 MYA 50 MYA 55 MYA

Epihippus

35 MYA

Miohippus

Oligocene

Mesohippus

25 MYA

Toe reduction The feet of modern horses have a single toe enclosed in a tough, bony hoof. By contrast, Hyracotherium had four toes on its front feet and three on its hind feet. Rather than hooves, these toes were encased in fleshy pads like those of dogs and cats. Examination of fossils clearly shows the transition through time: a general increase in length of the central toe, development of the bony hoof, and reduction and loss of the other toes (figure 21.12). As with body size, these trends occurred concurrently on several different branches of the horse evolutionary tree and were not exhibited by all lineages. At the same time that toe reduction was occurring, these horse lineages were evolving changes in the length and skeletal structure of their limbs, leading to animals capable of running long dis­tances at high speeds.

15 MYA 20 MYA

Changes in size The first species of horses were as big as a large house cat or a medium-sized dog. By contrast, modern equids can weigh more than 500 kg. Examination of the fossil record reveals that horses changed little in size for their first 30 million years, but since then, a number of different lineages have exhibited rapid and substantial increases. However, evolution has not been unidirectional and trends toward decreased size were also exhibited in some branches of the equid evolutionary tree, as revealed, for example, by Nannippus.

Anchitherium

10 MYA

Orohippus

The earliest known members of the horse family, species in the genus Hyracotherium, didn’t look much like modern-day horses at all. Small, with short legs and broad feet, these species occurred in wooded habitats, where they probably browsed on leaves and herbs and escaped predators by dodging through openings in the forest vegetation. The evolutionary path from these diminutive creatures to the workhorses of today has involved changes in a variety of traits, including size, toe reduction, and tooth size and shape (figure 21.12).

5 MYA

Hypohippus

The first horse

Megahippus

Pliocene

browsers grazers mixed feeders

Hyracotherium

well-documented case of how evolution has proceeded through adaptation to changing environments.

60 MYA

Hyracotherium (browsers)

Mesohippus (browsers)

Anchitherium (browsers)

Figure 21.12 Evolutionary change in body size of horses. Lines indicate evolutionary relationships of the horse family. Horse evolution is more like a bush than like a single-trunk tree; diversity was much greater in the past than it is today. In general, there has been a trend toward larger size, more complex molar teeth, and fewer toes, but this trend has exceptions. For example, a relatively recent form, Nannippus, evolved in the opposite direction, toward decreased size.

?

Inquiry question Why might the evolutionary line leading to Nannippus have experienced an evolutionary decrease in body size?

the same time, horses were eating grasses and other vegetation that contained more grit and other hard substances, thus favoring teeth better suited for withstanding such materials.

Evolutionary trends For many years, horse evolution was held up as an example of constant evolutionary change through time. Some even saw in the record of horse evolution evidence for a progressive, guiding force,

Neohipparion (grazers)

consistently pushing evolution in a single direction, toward longer limbs, fewer toes, and larger and more complex teeth. We now know that such views are misguided, and that the course of evolutionary change over millions of years is rarely so simple. Rather, the fossils demonstrate that even though overall trends have been evident in a variety of characteristics, evolutionary change has been far from constant and uniform through time. Instead, rates of evolution have varied widely, with long periods of little observable change and some periods of great change. Moreover, when changes happen, they often occur simultaneously in multiple lineages of the horse evolutionary tree. Finally, even when a trend exists, exceptions, such as the evolutionary decrease in body size exhibited by some lineages, are not uncommon. These patterns are usually discovered for any group of plants and animals for which we have an extensive fossil record, as you will see when we discuss human evolution in chapter 34.

Horse diversity One reason that horse evolution was originally conceived of as linear through time may be that modern horse diversity is relatively limited. For this reason it is easy to mentally picture a straight line from

Nannippus (grazers)

Equus

Dinohippus

Onohippidion

Astrohippus

Pliohippus

Calippus

Protohippus

Cormohipparion

Nannippus

Hipparion

Neohipparion

Pseudhipparion

Merychippus

Parahippus

Desmatippus

Archaeohippus

Kalobatippus

Merychippus (mixed feeders)

Equus (grazers)

Hyracotherium to modern-day Equus. But today’s limited horse diversity—only one surviving g­ enus—is unusual. In fact, at the peak of horse diversity in the Miocene epoch, 13 genera of horses could be found in North America alone. These species differed in body size and in a wide variety of other characteristics. Presumably, they lived in different habitats and exhibited different dietary preferences. Had this diversity existed to modern times, early evolutionary biologists would likely have had a different outlook on horse evolution.

Learning Outcomes Review 21.4

Fossils form when an organism is preserved in a matrix such as amber, permafrost, or rock. They can be used to construct a record of evolutionary transitions over long periods of time, which allows us to understand how major changes in evolution occur. The extensive fossil record for horses provides a detailed view of evolutionary diversification of this group, although trends are not constant and uniform and may include exceptions. ■■

Why might rates and direction of evolutionary change vary through time?

chapter

21 The Evidence for Evolution 453

should these very different structures be composed of the same bones—a single upper forearm bone, a pair of lower forearm bones, several small carpals, and one or more digits? If evolution had not occurred, this would indeed be a riddle. But when we consider that all of these animals are descended from a common ancestor, it is easy to understand that natural selection has modified the same initial starting blocks to serve very different purposes.

21.5 Anatomical

Evidence for Evolution

Learning Outcomes 1. Explain the evolutionary significance of homologous and vestigial structures 2. Describe how patterns of early development provide evidence for evolution.

Much of the power of the theory of evolution is its ability to provide a sensible framework for understanding the diversity of life. Many observations from throughout biology simply cannot be understood in any meaningful way except as a result of evolution.

Homologous structures suggest common derivation As vertebrates have evolved, the same bones have sometimes been put to different uses. Yet the bones are still recognizable, their presence betraying their evolutionary past. For example, the forelimbs of vertebrates are all homologous structures—structures with different appearances and functions that all derived from the same body part in a common ancestor. You can see in figure 21.13 how the bones of the forelimb have been modified in different ways for different mammals. Why

Early embryonic development shows similarities in some groups Some of the strongest anatomical evidence supporting evolution comes from comparisons of how organisms develop. Embryos of different types of vertebrates, for example, often are similar early on, but become more different as they develop. Early in their development, vertebrate embryos possess pharyngeal pouches, which develop into different structures. In humans, for example, they become various glands and ducts; in fish, they turn into gill slits. At a later stage, all primate embryos have a long tail, but whereas monkeys and most primates keep the tail, all we and our ape relatives retain is the coccyx at the end of our spine. Human fetuses even possess a fine fur (called lanugo) during the fifth month of development. Similarly, although most frogs go through a tadpole stage, some species develop directly and hatch out as little, fully formed frogs. However, the embryos of these species still exhibit tadpole features, such as the presence of a tail, which disappear before the froglet hatches (figure 21.14). These relict developmental forms suggest strongly that our development has evolved, with new instructions modifying ancestral developmental patterns. We have discussed the topic of embryonic development and evolution in chapter 19.

Some structures are imperfectly suited to their use

Humerus

Radius Ulna Carpals Metacarpals Phalanges Human

Cat

Bat

Porpoise

Horse

Figure 21.13 Homology of the bones of the forelimb of mammals. Although these structures show considerable differences in form and function, the same basic bones are present in the f­ orelimbs of humans, cats, bats, porpoises, and horses.

454 part IV Evolution

Because natural selection can work only on the variation present in a population, it should not be surprising that some organisms do not appear perfectly adapted to their environments. For example, most animals with long necks have many neck vertebrae for enhanced flexibility: geese have up to 25, and plesiosaurs, the long-necked reptiles that patrolled the seas during the age of dinosaurs, had as many as 76. By contrast, giraffes have only 7 very long neck vertebrae. Why haven’t they evolved more, like other long-necked animals? It turns out that almost all mammals have only 7 neck vertebrae. Because mammal species have no variation in vertebra number among individuals in a population, natural selection has nothing to work with, and thus there is no way for selection to lead to increased numbers of vertebrae (why other types of animals are more variable is a question for which we currently don’t have an answer). In the absence of variation in vertebra number, selection led to an evolutionary increase in vertebra size to produce the long neck of the giraffe. An excellent example of an imperfect design is the eye of vertebrate animals, in which the photoreceptors face backward, toward the wall of the eye (figure 21.15a). As a result, the nerve fibers extend not backward, toward the brain, but forward into the eye chamber, where they slightly obstruct light. Moreover,

Such examples illustrate that natural selection is like a tinkerer, working with whatever material is available to craft a workable solution, rather than like an engineer, who can design and build the best possible structure for a given task. Workable, but imperfect, structures such as the vertebrate eye are an expected outcome of evolution by natural selection.

Vestigial structures can be explained as holdovers from the past

Figure 21.14 Developmental features reflect evolutionary ancestry. Some species of frogs have lost the

tadpole stage. Nonetheless, tadpole features first appear and then disappear during development in the egg. ©James Hanken, Museum of

Comparative Zoology, Harvard University, Cambridge

these fibers bundle together to form the optic nerve, which exits through a hole at the back of the eye, creating a blind spot. By contrast, the eyes of mollusks—such as squid and ­octopuses—are better arranged: the photoreceptors face forward, and the nerve fibers exit at the back, neither obstructing light nor creating a blind spot (figure 21.15b).

Many organisms possess vestigial structures that have no apparent function, but resemble structures their ancestors possessed. Humans, for example, possess a complete set of muscles for wiggling their ears, just as many other mammals do. Although these muscles allow other mammals to move their ears to pinpoint sounds such as the movements or growl of a predator, they have little purpose in humans other than amusement. As other examples, boa constrictors have hip bones and rudimentary hind legs. Manatees (a type of aquatic mammal often referred to as “sea cows”) have fingernails on their flippers, which evolved from legs (figure 21.16). Some blind cave fish, which never see the light of day, have small, nonfunctional eyes (other blind cave fish have lost their eyes entirely). The human vermiform appendix has long been thought to be vestigial; it represents the degenerate terminal part of the cecum, the blind pouch or sac in which the large intestine begins. In other ­mammals, such as mice, the cecum is the largest part of the large intestine and functions in storage—usually of bulk cellulose in herbivores. Although greatly diminished in size, some recent ­ ­evidence suggests that the human appendix may not technically be vestigial because it may harbor beneficial intestinal bacteria. ­Vestigial or not, the human appendix can be a dangerous organ: appendicitis, which results from infection of the appendix, can be fatal. Vestigial traits can also be seen in the genomes of many organisms. For example, the icefish (figure 21.17) is a bizarrelooking, nearly see-through fish that lives in the frigid waters of the Antarctic. The icefish’s transparency results not only from a lack of pigment in its body structures, but also from the near invisibility of its blood. Our blood is red due to the presence of red

Blind spot Photoreceptor cells Interneuron

Photopigment Nerve impulse

a.

Photopigment

Nerve fibers

Light To brain via optic nerve

Photoreceptor cells

Light

Nerve fibers to brain

b.

Figure 21.15 The eyes of vertebrates and mollusks. a. Photoreceptors of vertebrates point backward, whereas (b) those of mollusks face forward. As a result, vertebrate nerve fibers pass in front of the photoreceptor—and where they bundle together and exit the eye, a blind spot is created. Mollusks’ eyes have neither of these problems. chapter

21 The Evidence for Evolution 455

Figure 21.16 Vestigial structures. The flippers of the West Indian manatee (Trichechus manatus), a relative of the elephant and descended from a terrestrial mammal, have retained nails, even though the manatee never leaves the water.

blood cells, which contain hemoglobin, the molecule that transports oxygen from the lungs to the tissues (see chapter 47). ­However, oxygen concentration in water increases as temperature decreases. The waters of the Antarctic, which are about 0°C, contain so much oxygen that the fish do not need special molecules to carry oxygen. The result is that these fish do not have hemoglobin, and consequently their blood is colorless. Nonetheless, when the DNA of icefish was examined, scientists discovered that they have the same gene that produces hemoglobin as that found in other vertebrates. However, the icefish hemoglobin gene has a variety of mutations that render it nonfunctional, and thus the icefish does not produce hemoglobin. The presence of this inoperative version of the hemoglobin gene in icefish means that its ancestors had hemoglobin; however, once the icefish’s progenitors occupied

the cold waters of the Antarctic and lost the need for hemoglobin, mutations that would prevent the production of hemoglobin and thus would be filtered out by natural selection in other species were able to persist in the population. Just by chance, some of these mutations increased in frequency in the population through time (the process of genetic drift, discussed in chapter 20), eventually becoming established in all individuals and knocking out the fish’s ability to produce hemoglobin. Pseudogenes, sometimes called fossil genes because they are traces of previously functioning genes, such as the hemoglobin gene in the icefish, are actually quite common in the genomes of most organisms and are discussed in chapter 24: when a trait disappears, the gene does not just vanish from the genome; rather, some mutation renders it inactive, and once that occurs, other mutations can accumulate. It is difficult to understand vestigial structures such as these as anything other than evolutionary relicts, holdovers from the past. However, the existence of vestigial structures argues strongly for the common ancestry of the members of the groups that share them, regardless of how different those groups have subsequently become. All of these anatomical lines of evidence—homology, development, and imperfect and vestigial structures—are readily understandable as a result of descent with modification, that is, evolution.

Learning Outcomes Review 21.5

Comparisons of the anatomy of different living animals often reveal evidence of shared ancestry. In cases of homology, the same organ has evolved to carry out different functions. In other cases, an organ is still present, usually in diminished form, even though it has lost its function altogether; such an organ or structure is termed vestigial. ■■

What are alternative explanations for homologous and vestigial structures?

21.6 Convergent

Evolution and the Biogeographical Record

Learning Outcomes 1. Explain the principle of convergent evolution. 2. Demonstrate how the biogeographical distribution of plant and animal species on islands provides evidence of evolutionary diversification.

Figure 21.17 Photo of an icefish. This nearly transparent fish, photographed from above, is found in the frigid waters of the Antarctic. British Antarctic Survey/Science Source 456 part IV Evolution

Biogeography, the study of the geographic distribution of species, reveals that different geographical areas sometimes exhibit

groups of plants and animals of strikingly similar appearance, even though the organisms may be only distantly related. It is difficult to explain so many similarities as the result of coincidence. Instead, natural selection appears to have favored parallel evolutionary adaptations in similar environments. Because selection in these instances has tended to favor changes that made the two groups more alike, their phenotypes have converged. This form of evolutionary change is referred to as convergent evolution.

Marsupials and placentals demonstrate convergence In the best-known case of convergent evolution, two major groups of mammals—marsupials and placentals—have evolved in very similar ways in different parts of the world. Marsupials are a group in which the young are born in a very immature condition and held in a pouch until they are ready to emerge into the outside world. In placentals, by contrast, offspring are not born until they can safely survive in the external environment (with varying degrees of parental care). Australia separated from the other continents more than 70 mya; at that time, both marsupials and placental mammals had evolved, but in different places. In particular, only marsupials occurred in Australia. As a result of this separation, the only placental mammals in Australia today are bats and a few colonizing rodents (which arrived relatively recently), and the continent is dominated by marsupials. What are the Australian marsupials like? To an astonishing degree, they resemble the placental mammals living today on the other continents (figure 21.18). The similarity between some individual members of these two sets of mammals argues strongly that they are the result of convergent evolution, similar forms having

Niche

Placental Mammals

Burrower

Anteater

Mole

Nocturnal Insectivore

Convergent evolution is a widespread phenomenon When species interact with the environment in similar ways, they often are exposed to similar selective pressures, and they therefore frequently develop the same evolutionary adaptations. Consider, for example, fast-moving marine predators (­figure 21.19). The hydrodynamics of moving through water require a streamlined body shape to minimize friction. It is no coincidence that dolphins, sharks, and tuna—among the fastest of marine ­species—have all evolved to have the same basic shape. We can infer as well that ichthyosaurs—marine reptiles that lived during the Age of the Dinosaurs—exhibited a similar lifestyle. Island trees exhibit a similar phenomenon. Most islands are covered by trees (or were until the arrival of humans). Careful inspection of these trees, however, reveals that they are not closely related to the trees with which we are familiar. Although they have all the characteristics of trees, such as being tall and having a tough outer covering, in many cases island trees are members of plant families that elsewhere exist only as flowers, shrubs, or other small bushes. For example, on many islands, the native trees are members of the sunflower family. Why do these plants evolve into trees on islands? Probably because seeds from trees rarely make it to isolated islands. As a result, those species that do manage to colonize distant islands face an empty ecological landscape upon arrival. In the absence of other treelike plants, natural selection often favors individual­plants that can capture the most sunlight for photosynthesis, and the result is the evolution of similar treelike forms on islands throughout the world.

Climber

Grasshopper mouse

Glider

Stalking Predator

Flying squirrel

Wolf

Numbat

Marsupial mole

Thylacine

Tree kangaroo Marsupial mouse

Chasing Predator

Ocelot

Ring-tailed lemur

Lesser anteater

Australian Marsupials

evolved in different, isolated areas because of similar selective pressures in similar environments.

Flying phalanger

Tasmanian quoll

Figure 21.18 Convergent evolution. Many marsupial species in Australia resemble placental mammals occupying similar ecological niches elsewhere in the rest of the world. Marsupials evolved in isolation after Australia separated from other continents. chapter

21 The Evidence for Evolution 457

Figure 21.19 Convergence among fast-swimming predators. Fast movement through water requires a streamlined body form,

which has evolved numerous times.

Convergent evolution is even seen in humans. People in most populations stop producing lactase, the enzyme that digests milk, sometime in childhood. However, individuals in African and European populations that raise cattle produce lactase throughout their lives. DNA analysis indicates that the retention of lactase production into adulthood is the result of different mutations in Africa and Europe, which indicates that the populations have independently (convergently) acquired this adaptation.

Biogeographical studies provide further evidence of evolution Darwin made several important observations during his voyage around the world. He noted that islands often are missing plants and animals common on continents, such as frogs and land mammals. Accidental human introductions have proved that these species can survive if they are released on islands, so lack of suitable habitat is not the cause. In addition, those species that are present on islands often have diverged from their continental relatives and sometimes—as with Darwin’s finches and the island trees just discussed—occupy ecological niches used by other species on continents. Lastly, island species usually are more closely related to species on nearby continents, even though the environment on continents and nearby islands often is not very similar. Darwin deduced the explanation for these phenomena. Many islands have never been connected to continental areas. The species that occur there arrived by dispersing across the water. Some species— those that can fly, float, or swim—are more likely to get to the island than others. Some, like frogs, are particularly vulnerable to dehydration in salt­water and have almost no chance of island colonization. The absence of some types of plants and animals provides opportunity to those that do arrive; as a result, colonizers, which usually come from nearby areas, often evolve into many species exhibiting great ecological and morph­ological diversity. This phenomenon, termed adaptive radiation, is discussed in chapter 22.

Learning Outcomes Review 21.6

Convergence is the evolution of similar forms in different lineages when exposed to similar selective pressures. The biogeographical distribution of species often reflects the outcome of evolutionary diversification with closely related species in nearby areas. ■■

Why does convergent evolution occur and why might species occupying similar environments in different localities sometimes not exhibit it?

458 part IV Evolution

21.7 Darwin’s

Critics

Learning Outcomes 1. Characterize the criticisms of evolutionary theory and list counterarguments that can be made. 2. Distinguish between hypothesis and theory in scientific usage.

In the century and a half since he proposed it, Darwin’s theory of evolution by natural selection has become nearly universally accepted by biologists, but has been a source of controversy among some members of the general public. Here we discuss seven principal objections that critics raise to the teaching of evolution as biological fact, along with some answers that scientists present in response: 1. Evolution is not solidly demonstrated. “Evolution is just a theory,” Darwin’s critics point out, as though theory meant a lack of knowledge, or some kind of guess. Scientists, however, use the word theory in a very different sense than the general public does. They use theory only for those ideas that are strongly supported by many lines of evidence, like the theories of gravity and evolution. It is important to recognize that both evolution and gravitation are “just theories.” The term theory is not the equivalent of a “hunch” or a “notion,” or even an initial hypothesis. Rather, it is a well-tested phenomenon that rationalizes the data available. 2. There are no fossil intermediates. “No one ever saw a fin on the way to becoming a leg,” critics claim, pointing to the many gaps in the fossil record in Darwin’s day. Since that time, however, many fossil intermediates in vertebrate evolution have indeed been found. A clear line of fossils now traces the transition between hoofed mammals and whales, between reptiles and mammals, between dinosaurs and birds, and between apes and humans. The fossil evidence of evolution from one major type to another is compelling. 3. The intelligent design argument. “The organs of living creatures are too complex for a random process to have produced them—the existence of a clock is evidence of the existence of a clockmaker.” Evolution by natural selection is not a random process. Quite the contrary, by favoring those variations

that lead to the highest reproductive fitness, natural selection is a nonrandom process that can construct highly complex organs by incrementally improving them from one generation to the next. For example, the intermediates in the evolution of the mammalian ear can be seen in fossils, and many intermediate “eyes” are known in various invertebrates. These intermediate forms arose because they have value—being able to detect light slightly is better than not being able to detect it at all. Complex structures such as eyes evolved as a progression of slight improvements. Moreover, inefficiencies of certain designs, such as the vertebrate eye and the existence of vestigial structures, do not support the idea of an intelligent designer. 4. Evolution violates the Second Law of Thermodynamics. “A jumble of soda cans doesn’t by itself jump neatly into a stack—things become more disorganized due to random events, not more organized.” Biologists point out that this argument ignores what the second law really says: disorder increases in a closed system, which the Earth most certainly is not. Energy continually enters the biosphere from the Sun, fueling life and all the processes that organize it. 5. Proteins are too improbable. “Hemoglobin has 141 amino acids. The probability that the first one would be leucine is 1/20, and that all 141 would be the ones they are by chance is (1/20)141, an impossibly rare event.” This argument illustrates a lack of understanding of probability and statistics—probability cannot be used to argue backward. The probability that a student in a classroom has a particular birthdate is 1/365; arguing this way, the probability that everyone in a class of 50 would have the birthdates that they do is (1/365)50, and yet there the class sits, all with their actual birthdates. 6. Natural selection cannot account for major ­changes in evolution. “No scientist has come up with an experiment in which fish evolve into frogs and leap away from predators.” Can we extrapolate from our understanding that natural selection produces relatively small changes that are observable in populations within species to explain the major differences observed between species? Most biologists who have studied the problem think so. The differences between breeds produced by artificial selection—such as chihuahuas, mastiffs, and greyhounds—are more distinctive than the differences between some wild species, and laboratory selection experiments sometimes create forms that cannot interbreed and thus would in nature be considered different species. Thus, production of radically different forms has indeed been observed, repeatedly. These changes usually take millions of years, and they are seen clearly in the fossil record. 7. The irreducible complexity argument. Because each part of a complex cellular mechanism such as blood clotting is essential to the overall process, the intricate machinery of the cell cannot be explained by evolution from simpler stages. Without all of the parts functioning, the system would not work. Consequently, evolution couldn’t have

built the structure because intermediate stages, with not all parts in place, would not function and thus would not be favored by natural selection. What’s wrong with this argument is that each part of a complex molecular machine evolves as part of the whole system. Natural selection can act on a complex system because at every stage of its evolution, the system functions. Parts that improve function are added. Subsequently, other parts may be modified or even lost, so that parts that were not essential when they first evolved become essential. In this way, an “irreducibly complex” structure can evolve by natural selection. The same process works at the molecular level. For example, snake venom initially evolved as enzymes to increase the ability of snakes to digest large prey items, which were captured by biting the prey and then constricting them with coils. Subsequently, the digestive enzymes evolved to become increasingly lethal. Rattlesnakes kill large prey by injecting them with venom, letting them go, and then tracking them down and eating them after they die. To do so, they have evolved extremely toxic venom, highly modified syringe-like front teeth, and many other characteristics. Take away the fangs or the venom and the rattlesnakes can’t feed—what initially evolved as nonessential parts are now indispensable; irreducible complexity has evolved by natural selection. The mammalian blood clotting system similarly has evolved from much simpler systems. The core clotting system evolved at the dawn of the vertebrates more than 500 mya, and it is found today in primitive fishes such as lampreys. One hundred million years later, as vertebrates continued to evolve, proteins were added to the clotting system, making it sensitive to substances released from damaged tissues. Fifty million years later, a third component was added, triggering clotting by contact with the jagged surfaces produced by injury. At each stage, as the clotting system evolved to become more complex, its overall performance came to depend on the added elements. Thus, blood clotting has become “irreducibly complex” as the result of Darwinian evolution. Statements that various structures could not have been built by natural selection have repeatedly been made over the past 150 years. In many cases, after detailed scientific study, the likely path by which such structures have evolved has been discovered.

Learning Outcomes Review 21.7

Darwin’s theory of evolution is controversial to some in the general public. Objections are often based on a misunderstanding of the theory. In scientific usage, a hypothesis is an educated guess, whereas a theory is an explanation that fits available evidence and has withstood rigorous testing. ■■

Suppose someone suggested that humans originally came from Mars. Would this be a hypothesis or a theory, and how could it be tested? chapter

21 The Evidence for Evolution 459

Chapter Review Georgia Southern University Museum

21.1 The Beaks of Darwin’s Finches: Evidence

of Natural Selection

Galápagos finches exhibit variation related to food gathering. The correspondence between beak shape and its use in obtaining food suggested to Darwin that finch species had diversified and adapted to eat different foods.

Fossils present a history of evolutionary change. Fossils document evolutionary transitions. The history of life on Earth can be traced through the fossil record. In recent years, new fossil discoveries have provided more detailed understanding of major evolutionary transitions.

Modern research has verified Darwin’s selection hypothesis. Natural selection acts on variation in beak morphology, favoring largerbeaked birds during extended droughts and smaller-beaked birds during long periods of heavy rains. Because this variation is heritable, evolutionary change occurs in the frequencies of beak sizes in subsequent generations.

The evolution of horses is a prime example of evidence from fossils. The fossil record indicates that horses have evolved from small, forestdwelling animals to the large and fast plains-dwelling species alive today. Over the course of 50 million years, evolution has not been constant and uniform. Rather, change has been rapid at some times, slow at others. Although a general trend toward increase in size is evident, some species evolved to smaller sizes.

21.2 Peppered Moths and Industrial Melanism: More

21.5 Anatomical Evidence for Evolution

Evidence of Selection

Light-colored moths decreased in polluted areas. In polluted areas where soot built up on tree trunks, the dark-colored form of the peppered moth became more common. In unpolluted areas, light-colored forms remained predominant. Experiments suggested that predation by birds was the cause; lightcolored moths stand out on dark trunks, and vice versa. When environmental conditions reverse, so does selection pressure. In the last 50 years, pollution has decreased in many areas and the frequency of light-colored moths has rebounded. The agent of selection may be difficult to pin down. Some have questioned whether bird predation is the agent of selection, but recent research supports this hypothesis. Regardless, the observation that the dark-colored form has increased during times of pollution and then declined as pollution abates indicates that natural selection has acted on moth coloration.

Homologous structures suggest common derivation. Homologous structures may have different appearances and functions even though derived from the same common ancestral body part. Early embryonic development shows similarities in some groups. Embryonic development shows similarity in developmental patterns among species whose adult phenotypes are very different. Species that have lost a feature that was present in an ancestral form often develop and then lose that feature during embryological development. Some structures are imperfectly suited to their use. Natural selection can influence only the variation present in a population; because of this, evolution often results in workable, but imperfect, structures, such as the vertebrate eye. Vestigial structures can be explained as holdovers from the past. The existence of vestigial structures supports the concept of common ancestry among organisms that share them.

21.3 Artificial Selection: Human-Initiated Change

21.6 Convergent Evolution and the

Experimental selection produces changes in populations. Laboratory experiments in directional selection have shown that substantial evolutionary change can occur in these controlled populations.

Marsupials and placentals demonstrate convergence. Convergent evolution may occur in species or populations exposed to similar selective pressures. Marsupial mammals in Australia have converged upon features of their placental counterparts elsewhere.

Agricultural selection has led to extensive modification of crops and livestock.

Convergent evolution is a widespread phenomenon. Examples include hydrodynamic streamlining in marine species and the evolution of tree species on islands from ancestral forms that were not treelike.

(figure 21.5)

Domesticated breeds have arisen from artificial selection. Crop plants and domesticated animal breeds are often substantially different from their wild ancestors. If artificial selection can rapidly create substantial change over short periods of time, then it is reasonable to assume that natural selection could have created the Earth’s diversity of life over millions of years.

21.4 Fossil Evidence of Evolution The age of fossils can be estimated. Specimens become fossilized in different ways. Fossils in rock can be dated by calculating the extent of radioactive decay based on half-lives of known isotopes.

460 part IV Evolution

Biogeographical Record

Biogeographical studies provide further evidence of evolution. Island species usually are closely related to species on nearby continents even if the environments are different. Early island colonizers often evolve into diverse species because other, competing species are scarce.

21.7 Darwin’s Critics Darwin’s theory of evolution by natural selection is almost universally accepted by biologists. Many criticisms have been made both historically and recently, but most stem from a lack of understanding of scientific principles, the theory’s actual content, or the time spans involved in evolution.

Visual Summary Evidence for evolution includes evidence from

Natural selection

Artificial selection

Fossils

exemplified by

Peppered moths

Transitional species

exemplified by

exemplified by

Crops

Livestock

Convergent evolution

Biogeographical record

seen in

include

used to modify Darwin’s finches

Anatomy

Marsupials and placental mammals

revealed through

Horses

differ from Wild ancestors

Homologous structures

Embryological development

Imperfect structures

Vestigial structures

Island vs. mainland species

Review Questions Georgia Southern University Museum

U N D E R S TA N D 1. Artificial selection is different from natural selection because a. b. c. d.

artificial selection is not capable of producing large changes. artificial selection does not require genetic variation. natural selection cannot produce new species. breeders (people) choose which individuals reproduce based on desirability of traits.

2. Gaps in the fossil record a. b. c. d.

demonstrate our inability to date geological sediments. are expected since the probability that any organism will fossilize is extremely low. have not been filled in as new fossils have been discovered. weaken the theory of evolution.

3. The evolution of modern horses (Equus) is best described as a. b. c. d.

the constant change and replacement of one species by another over time. a complex history of lineages that changed over time, with many going extinct. a simple history of lineages that have always resembled extant horses. None of the choices is correct.

4. Homologous structures a. b. c. d.

are structures in two or more species that originate as the same structure in a common ancestor. are structures that look the same in different species. cannot serve different functions in different species. must serve different functions in different species.

5. Convergent evolution a. b. c. d.

is an example of stabilizing selection. depends on natural selection to independently produce similar phenotypic responses in different species or populations. occurs only on islands. is expected when different lineages are exposed to vastly different selective environments.

6. Darwin’s finches are a noteworthy case study of evolution by natural selection because evidence suggests a. b. c. d.

they are descendants of many different species that colonized the Galápagos. they radiated from a single species that colonized the Galápagos. they are more closely related to mainland species than to one another. None of the choices is correct. chapter

21 The Evidence for Evolution 461

7. The possession of fine fur in 5-month human embryos indicates a. b. c. d.

that the womb is cold at that point in pregnancy. that humans evolved from a hairy ancestor. that hair is a defining feature of mammals. that some parts of the embryo grow faster than others.

A P P LY 1. In Darwin’s finches, a. b. c. d.

occurrence of wet and dry years preserves genetic variation for beak size. increasing beak size over time proves that beak size is inherited. large beak size is always favored. All of the choices are correct.

2. Artificial selection experiments in the laboratory such as in figure 21.5 are an example of a. b. c. d.

stabilizing selection. negative frequency-dependent selection. directional selection. disruptive selection.

3. Convergent evolution is often seen among species on different islands because a. b. c. d.

island populations are usually smaller and more affected by genetic drift. disruptive selection occurs commonly on islands. island species are usually most closely related to species in similar habitats elsewhere. when islands are first colonized, many ecological resources are unused, allowing descendants of a colonizing species to diversify and adapt to many different parts of the environment.

462 part IV Evolution

SYNTHESIZE 1. What conditions are necessary for evolution by natural selection? 2. Explain how data shown in figure 21.2a and b relate to the conditions identified by you in question 1. 3. On figure 21.2b, draw the relationship between offspring beak depth and parent beak depth, assuming that there is no genetic basis to beak depth in the medium ground finch. 4. Refer to figure 21.5, artificial selection in the laboratory. In this experiment, one population of Drosophila was selected for low numbers of bristles and the other for high numbers. Note that not only did the means of the populations change greatly in 35 generations, but also all individuals in both experimental populations lie outside the range of the initial population. What would happen if the direction of selection were reversed, such that a greater number of bristles was selected for in the low-bristle population, and vice versa? How would the rate of evolutionary change compare with that in the initial part of the experiment before selection was reversed? 5. The ancestor of horses was a small, many-toed animal that lived in forests, whereas today’s horses are large animals with a single hoof that live on open plains. A series of intermediate fossils illustrate how this transition has occurred, and for this reason, many old treatments of horse evolution portrayed it as a steady increase through time in body size accompanied by a steady decrease in toe number. Why is this interpretation incorrect?

22 CHAPTER

The Origin of Species Chapter Contents 22.1

The Nature of Species and the Biological Species Concept

22.2 Natural Selection and Reproductive Isolation 22.3 The Role of Genetic Drift and Natural Selection in Speciation 22.4 The Geography of Speciation 22.5 Adaptive Radiation and Biological Diversity 22.6 The Pace of Evolution 22.7 Speciation and Extinction Through Time

JohnMernick/iStock/Getty Images

Introduction

Visual Outline defined by

Biological species concept

Geography

requires

affects

Reproductive isolation

can be shaped by

leads to

affects

Speciation

Natural selection

may affect

Genetic drift

resulting in

Fluctuations in species numbers

can lead to

PACIFIC OCEAN

Evolutionary diversification

1000

NEW GUINEA

producing

Adaptive radiation

Number of families

Species

800 600 Cretaceous 400 200 0 600

Devonian Ordovician

500

Permian Triassic

400 300 200 Millions of years ago

100

0

Although Darwin titled his book On the Origin of Species, he never actually discussed what he referred to as that “mystery of mysteries”— how one species gives rise to another. Rather, his argument concerned evolution by natural selection—that is, how one species evolves through time to adapt to its changing environment. Although an important mechanism of evolutionary change, the process of adaptation does not explain how one species becomes another, a process we call speciation. As we shall see, adaptation may be involved in the speciation process, but it does not have to be. Before we can discuss how one species gives rise

to another, we need to understand exactly what a species is. Even though the definition of a species is of fundamental importance to evolutionary biology, this issue has still not been completely settled and is currently the subject of considerable research and debate.

22.1

The Nature of Species and the Biological Species Concept

Black Intergrade

Yellow

Learning Outcomes 1. Understand the biological species concept and why it does not explain all observations. 2. Define the two kinds of reproductive isolating mechanisms. 3. Describe the relationship of reproductive isolating mechanisms to the biological species concept.

Gray

Figure 22.1 Geographic variation in the eastern rat snake, Pantherophis alleghaniensis. Although populations at

the eastern, western, and northern ends of the species’ range are phenotypically quite distinctive from one another, they are connected by populations, labelled “intergrade,” that are phenotypically intermediate.

Any concept of a species must account for two phenomena: the distinctiveness of species that occur together at a single locality, and the connection that exists among different populations belonging to the same species.

distant populations may appear distinct, they are usually connected by intervening populations that are intermediate in their characteristics.

Sympatric species inhabit the same locale but remain distinct

The biological species concept focuses on the ability to exchange genes

Put out a birdfeeder on your balcony or in your back yard, and you will attract a wide variety of birds (especially if you include different kinds of foods). In the midwestern United States, for example, you might routinely see cardinals, blue jays, downy woodpeckers, house finches—even hummingbirds in the summer. Although it might take a few days of careful observation, you would soon be able to readily distinguish the many different species. The reason is that species that occur together (termed sympatric) are distinctive entities that are phenotypically different, utilize different parts of the habitat, and behave differently. This observation is generally true not only for birds, but also for most other types of organisms. Occasionally, two species occur together that appear to be nearly identical. In such cases, we need to go beyond visual similarities. When other aspects of the phenotype are examined, such as the mating calls or the chemicals exuded by each species, they usually reveal great differences. In other words, even though we might have trouble distinguishing them, the organisms themselves have no such difficulties.

Populations of a species exhibit geographic variation Within a single species, individuals in populations that occur in different areas may be distinct from one another. In areas where these populations occur close to each other, individuals often exhibit combinations of features characteristic of both populations (figure 22.1). In other words, even though geographically 464 part IV Evolution

What can account for both the distinctiveness of sympatric species and the connectedness of geographically separate populations of the same species? One obvious possibility is that each species exchanges genetic material only with other members of its species. If sympatric species commonly exchanged genes, which they generally do not, we might expect such species to rapidly lose their distinctions, as the gene pools (that is, all of the alleles present in a species) of the different species became homogenized. Conversely, the ability of geographically distant populations of a single species to share genes through the process of gene flow may keep these populations integrated as members of the same species. Based on these ideas, in 1942 the evolutionary biologist Ernst Mayr set forth the biological species concept, which defines species as “. . . groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups.” In other words, the biological species concept says that a species is composed of populations whose members mate with each other and produce fertile offspring—or would do so if they came into contact. Conversely, populations whose members do not mate with each other or who cannot produce fertile offspring are said to be reproductively isolated and, therefore, are members of different species. What causes reproductive isolation? If organisms cannot interbreed or cannot produce fertile offspring, they clearly ­belong to different species. However, some populations that are considered separate species can interbreed and produce fertile offspring, but they ordinarily do not do so under natural conditions. They are still

considered reproductively isolated because genes from one species generally will not enter the gene pool of the other. Table 22.1 summarizes the steps at which barriers to successful reproduction may occur. Such barriers are termed reproductive isolating mechanisms because they prevent gen­etic exchange between species. We will discuss examples of these next, beginning with those that prevent the formation of zygotes, which are called prezygotic isolating mechanisms. Mechanisms that prevent the proper functioning of zygotes after they form are called postzygotic isolating mechanisms.

TA B L E 2 2 .1 Mechanism

Reproductive Isolating Mechanisms Description

PRE Z YG OTI C I SO L ATI N G M E CH A N I S M S Ecological isolation

Species occur in the same area, but they occupy different habitats and rarely encounter each other.

Behavioral isolation

Species differ in their mating rituals.

Temporal isolation

Species reproduce in different seasons or at different times of the day.

Mechanical isolation

Structural differences between species prevent mating.

Prevention of gamete fusion

Gametes of one species function poorly with the gametes of another species or within the reproductive tract of another species.

Prezygotic isolating mechanisms prevent the formation of a zygote Mechanisms that prevent formation of a zygote include ecological or environmental isolation, behavioral isolation, temporal isolation, mechanical isolation, and prevention of gamete fusion.

Ecological isolation Even if two species occur in the same area, they may utilize different portions of the environment and thus not hybridize because they do not encounter each other. For example, lions and tigers can produce hybrid offspring in zoos (figure 22.2), but even though the ranges of the two species overlapped in India until about 150 years ago, no natural hybrids were ever discovered. Lions stayed mainly in the open grassland and hunted in groups called prides; tigers tended to be solitary creatures of the forest. Because of their ecological and behavioral differences, lions and tigers rarely came into direct contact with each other, even though their ranges overlapped over thousands of square kilometers. In another example, the ranges of two toads, Bufo wood­ housei and B. americanus, overlap in some areas. Although these two species can produce viable hybrids, they usually do not interbreed because they utilize different portions of the habitat for breeding. B. woodhousei prefers to breed in streams, and B. ameri­ canus breeds in rainwater puddles. Similar situations occur among plants. Two species of oaks occur widely in California: the valley oak, Quercus lobata, and the scrub oak, Q. dumosa. The valley oak, a graceful deciduous tree that can be as tall as 35 m, occurs in the fertile soils of open grassland on gentle slopes and valley floors. In contrast, the scrub oak is an evergreen shrub, usually only 1 to 3 m tall, which often forms the kind of dense scrub known as chaparral. The scrub oak is found on steep slopes in less fertile soils. Hybrids between these different oaks do occur and are fully fertile, but they are rare.

POSTZYGOTIC I SO L ATI N G M E CH A N I S M S Hybrid inviability or infertility

Hybrid embryos do not develop properly, hybrid adults do not survive in nature, or hybrid adults are sterile or have reduced fertility.

Figure 22.2 Lions and tigers are ecologically isolated. 

The ranges of lions and tigers overlap in India. However, lions and tigers do not hybridize in the wild because they utilize different portions of the habitat. Hybrids, such as this tiglon, have been successfully produced in captivity, but hybridization does not occur in the wild. Alexander Bayburov/Shutterstock chapter

22 The Origin of Species 465

Amplitude (dB)

Chrysoperla plorabunda

Chrysoperla adamsi

Chrysoperla johnsoni 0

1

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5

6

7

8

9

10

11

12

Time (seconds)

Figure 22.3 Differences in courtship rituals can isolate related bird species. These Gala ́pagos blue-footed boobies select their mates only after an elaborate courtship display. This male is lifting his feet in a ritualized high-step that shows off his bright blue feet. The display behavior of the two other species of boobies that occur in the Gala ́pagos is very different, as is the color of their feet.

Rene Baars/Shutterstock

The sharply distinct habitats of their parents limit their occurrence together, and there is little intermediate habitat where the hybrids might flourish.

Behavioral isolation Chapter 53 describes the often elaborate courtship and mating rituals of some groups of animals. Related species of organisms such as birds often differ in their courtship rituals, which tends to keep these species distinct in nature even if they inhabit the same places (figure 22.3). Sympatric species avoid mating with members of the wrong species in a variety of ways; every mode of communication imaginable appears to be used by some species. Differences in visual signals, as just discussed, are common; however, other types of animals rely more on other sensory modes for communication. Many species, such as frogs, birds, and a variety of insects, use sound to attract mates. Predictably, sympatric species of these animals produce different calls. Similarly, the “songs” of lacewings are produced when they vibrate their abdomens against the surface on which they are sitting, and sympatric species produce different vibration patterns (figure 22.4). Other species rely on the detection of chemical signals, called pheromones. The use of pheromones in moths has been particularly well studied. When female moths are ready to mate, they emit a pheromone that males can detect at great distances. Sympatric species differ in the pheromone they produce: either they use different chemical compounds, or, if they are using the same compounds, the proportions used are different. Laboratory studies indicate that males are remarkably adept at distinguishing the pheromones of their own species from those of other species or even from synthetic compounds similar, but not identical, to that of their own species. 466 part IV Evolution

Figure 22.4 Differences in courtship song of sympatric species of lacewings. Lacewings are small insects that rely on signals produced by moving their abdomens to vibrate the surface on which they are sitting to attract mates. As these recordings indicate, the vibration patterns produced by sympatric species differ greatly. Females, which detect the calls as they are transmitted through solid surfaces such as branches, are able to distinguish calls of different species and respond only to individuals producing their own species’ call.

Some species even use electroreception. African and South Asian electric fish independently have evolved specialized organs in their tails that produce electrical discharges and electroreceptors on their skins to detect them. These discharges are used to communicate in social interactions; field experiments indicate that males can distinguish between signals produced by their own and other, co-occurring species, probably on the basis of differences in the timing of the electrical pulses.

Temporal isolation Two species of wild lettuce, Lactuca graminifolia and L. cana­ densis, grow together along roadsides throughout the southeastern United States. Hybrids between these two species are easily made experimentally and are completely fertile. But these hybrids are rare in nature because L. graminifolia flowers in early spring and L. canadensis flowers in summer. When their blooming periods overlap, as happens occasionally, the two species do form hybrids, which may become locally abundant. Many species of closely related amphibians have different breeding seasons that prevent hybridization. For example, five species of frogs of the genus Rana occur together in most of the eastern United States, but hybrids are rare because the peak breeding time is different for each of them.

Mechanical isolation Structural differences prevent mating between some related species of animals. Aside from such obvious features as size, the

structures of the male and female copulatory organs may be incompatible. In many insect and other arthropod groups, the sexual organs, particularly those of the male, are so diverse that they are used as a primary basis for distinguishing species. Similarly, flowers of related species of plants often differ significantly in their proportions and structures. Some of these differences limit the transfer of pollen from one plant species to another. For example, bees may carry the pollen of one species on a certain place on their bodies; if this area does not come into ­contact with the receptive structures of the flowers of another plant species, the pollen is not transferred.

1

2

3

Prevention of gamete fusion In animals that shed gametes directly into water, the eggs and sperm derived from different species may not attract or fuse with one another. Many animals that have internal fertilization may not hybridize successfully because the sperm of one species functions so poorly within the reproductive tract of another that fertilization never takes place. In plants, the growth of pollen tubes may be impeded in hybrids between different species. In both plants and animals, isolating mechanisms such as these prevent the union of gametes, even following successful mating.

Postzygotic isolating mechanisms prevent normal development into reproducing adults All of the factors we have discussed so far tend to prevent hybridization. If hybrid matings do occur and zygotes are produced, many factors may still prevent those zygotes from developing into normally functioning, fertile individuals. As you saw in chapter 19, development is a complex process. In hybrids, the genetic complements of two species may be so different that they cannot function together normally in embryonic development. For example, hybridization between sheep and goats usually produces embryos that die in the earliest developmental stages. The leopard frogs (Rana pipiens complex) of the eastern United States are a group of similar species, assumed for a long time to constitute a single species (figure 22.5). Careful examination, however, revealed that although the frogs appear similar, successful mating between them is rare because of problems that occur as the fertilized eggs develop. Many of the hybrid combinations cannot be produced even in the laboratory. Examples of this kind, in which the existence of multiple species has been recognized only as a result of hybridization experiments, are common in plants. Even when hybrids survive the embryo stage, they may still not develop normally. If the hybrids are less physically fit than their parents, they will almost certainly be eliminated in nature. Even if a hybrid is vigorous and strong, as in the case of the mule, which is a hybrid between a female horse and a male donkey, it may still be sterile and thus incapable of contributing to succeeding generations. Hybrids may be sterile because the development of sex organs is abnormal, because the chromosomes derived from the respective parents cannot pair properly during meiosis, or due to a variety of other causes. In the case of the mule, for example, the parental species have different numbers of chromosomes, and as a result, the mule’s chromosomes cannot align properly during meiosis, thus rendering the animal infertile.

4

1. Rana pipiens 2. Rana blairi 3. Rana sphenocephala 4. Rana berlandieri

Figure 22.5 Postzygotic isolation in leopard frogs. 

These four species resemble one another closely in their external features. Their status as separate species first was suspected when hybrids between some pairs of these species were found to produce defective embryos in the laboratory. Subsequent research revealed that the mating calls of the four species differ substantially, indicating that the species have both pre- and postzygotic isolating mechanisms.

?

Inquiry question Can more than one type of reproductive isolating mechanism be operating between two species?

The biological species concept does not explain all observations The biological species concept has proved to be an effective way of understanding the existence of species in nature. Nonetheless, it fails to take into account all observations, leading some biologists to propose alternative species concepts. One criticism of the biological species concept concerns the extent to which all species truly are reproductively isolated. By definition, under the biological species concept, species should not interbreed and produce fertile offspring. But in recent years, biologists have detected much greater amounts of interspecific (i.e., between different species) hybridization than was previously thought to occur between populations that seem to coexist as distinct biological entities. Botanists have always been aware that plant species often undergo substantial amounts of hybridization. More than 50% of California plant species included in one study, for example, were not well defined by genetic isolation. This coexistence without genetic isolation can be long-lasting: fossil data show that balsam poplars and cottonwoods have been phenotypically distinct for 12 million years, but they also have routinely produced hybrids throughout this chapter

22 The Origin of Species 467

time. Consequently, many botanists have long felt that the biological species concept is misnamed and applies only to animals. New evidence, however, increasingly indicates that hybridization is not all that uncommon in animals, either. In recent years, many cases of substantial hybridization between animal species have been documented. One survey indicated that almost 10% of the world’s more than 10,000 bird species are known to have hybridized in nature. The Galápagos finches provide a particularly well-­studied example. Three species on the island of Daphne Major—the medium­ground finch, the cactus finch, and the small ground finch—are clearly distinct morphologically, and they occupy different ecological niches. Studies over the past 30 years by Peter and Rosemary Grant found that, on average, 2% of the medium ground finches and 1% of the cactus ground finches mated with other species every year. Furthermore, hybrid offspring appeared to be at no disadvantage in terms of survival or subsequent reproduction. This is not a trivial amount of genetic exchange, and one might expect to see the species coalesce into one genetically variable population—but the species are maintaining their distinctiveness. Hybridization is not rampant throughout the animal world, however. Most bird species do not hybridize, and probably even fewer experience significant amounts of hybridization. Still, hybridization is common enough to cast doubt on whether reproductive isolation is the only force maintaining the integrity of species.

Natural selection and the maintenance of species’ differences An alternative species concept, the ecological species concept, proposes that the distinctions among species are maintained by natural selection. The idea is that each species has adapted to its own specific part of the environment. Stabilizing selection, described in chapter 20, then maintains the species’ different adaptations. Hybridization has little ­effect because alleles introduced into one species’ gene pool from other species are quickly eliminated by natural selection. You probably recall from chapter 20 that the interaction between gene flow and natural selection can have many outcomes. In some cases, strong selection can overwhelm any effects of gene flow—this is how natural selection can maintain species’ differences in the face of gene flow. But, in other situations, gene flow can prevent natural selection from eliminating less successful alleles from a population; in this case, the biological species concept would be applicable.

Other weaknesses of the biological species concept The biological species concept has been criticized for other reasons as well. For example, it can be difficult to apply the concept to populations that are geographically separated in nature. Because individuals of these populations do not encounter each other, it is not possible to observe whether they would interbreed naturally. Although experiments can determine whether fertile hybrids can be produced, this information is not enough. Many species that coexist without interbreeding in nature will readily hybridize in the artificial settings of the laboratory or zoo. Consequently, evaluating whether such populations constitute different species is ultimately a judgment call. In addition, the concept is more limited than its name would imply. Many organisms are asexual and 468 part IV Evolution

reproduce without mating. Reproductive isolation therefore has no meaning for such organisms. For these reasons, a variety of other ideas have been put forward to establish criteria for defining species. Many of these are specific to a particular type of organism, and none has universal applicability. In reality, there may be no single explanation for what maintains the identity of species. Given the incredible variation evident in plants, animals, and microorganisms in all aspects of their biology, it would not be surprising to find that different processes are operating in different organisms. In addition, some scientists have turned from emphasizing the processes that maintain species distinctions to examining the evolutionary history of populations. These genealogical species concepts are currently a topic of great debate and are discussed further in chapter 23.

Learning Outcomes Review 22.1

Species are populations of organisms that are distinct from other, co-occurring species, and are interconnected geographically. The biological species concept therefore defines species based on their ability to interbreed. Reproductive isolating mechanisms prevent successful interbreeding between species. An alternative approach emphasizes the role of adaptation and natural selection as a force for maintaining separation of species. ■■

■■

How does the ability to exchange genes explain why sympatric species remain distinct and geographic populations of one species remain connected? How could natural selection explain this phenomenon?

22.2 Natural

Selection and Reproductive Isolation

Learning Outcomes 1. Define reinforcement in the context of reproductive isolation. 2. Explain the possible outcomes when two populations that are partially reproductively isolated become sympatric.

One of the oldest questions in the field of evolution is: How does one ancestral species become divided into two descendant species? If species are defined by the existence of reproductive isolation, then the process of speciation is identical to the evolution of reproductive isolating mechanisms.

Selection may reinforce isolating mechanisms The formation of species is a continuous process, and as a result, two populations may have gone only part of the way toward becoming different species and thus be only partially reproductively isolated. For example, because of behavioral or ecological differences, individuals of two populations may be more likely to mate with members of their own population, but ­between-population matings may still occur. If mating occurs and fertilization produces

a zygote, postzygotic barriers may also be incomplete: developmental problems may result in lower embryo survival or reduced fertility, but some individuals may survive and ­reproduce.

Pointed phlox Drummond’s phlox in sympatry Drummond’s phlox in allopatry

How reinforcement can complete the speciation process What happens when two populations come into contact thus depends on the extent to which isolating mechanisms have already evolved. If isolating mechanisms have not evolved at all, then the two populations will interbreed freely, and whatever differences have evolved between them should disappear over the course of time as genetic exchange homogenizes the populations. Conversely, if the populations are completely reproductively isolated, then no genetic exchange will occur and the two populations will remain different species. More interesting is the situation in which hybrids are produced that are either only partly sterile or not as well adapted to the existing habitats as their parents. Selection would favor any alleles in the parental populations that prevented hybridization, because individuals that did not engage in hybridization would produce more successful offspring. The result would be that selection would improve prezygotic isolating mechanisms until the two populations were completely reproductively isolated. This process is termed r­ einforcement because initially incomplete isolating mechanisms are reinforced by natural selection until they are completely effective. An example of reinforcement is provided by wildflowers called phlox (figure 22.6). Most phlox flowers are a pale blue color, sometimes with a pink tinge. Drummond’s phlox, a beautiful species that grows profusely along highways, is no exception. The one exception is in certain parts of Texas, where it is sympatric with another species, the pointed phlox. When the two species are found together, the pointed phlox maintains the pale blue coloration, but Drummond’s phlox is a deep shade of red. How has this color difference evolved, and why? The color of these flowers is a result of red and blue color pigments, produced by different genes and leading to the light blue, slightly pinkish color, and another gene that affects the amount of all pigments produced, affecting the intensity of the color. In sympatric populations of Drummond’s phlox, an allele that causes an increase in pigment production has replaced the normal allele. By itself, this change would lead to increased production of both red and blue pigments, leading to a much deeper shade of the same color. However, a new allele at a second gene has knocked out production of the blue pigments, leaving the red pigment to predominate. The result is the deep red color of the sympatric Drummond’s phlox flowers. But why has this change occurred? The answer lies in the interaction between the two phlox species. The flowers are able to interbreed, but their offspring have very low fertility. Consequently, any factor that would diminish interbreeding would be favored by natural selection. And that’s just what color does. Both species are pollinated by butterflies that go from one flower to another, and it turns out that individual butterflies have color preferences. As a result, some butterflies visit only the light blue flowers and others only the dark red ones, and thus pollen from one species does not fertilize flowers of the other species. This example illustrates how reproductive isolation initially resulting from postzygotic infertility can be reinforced by the evolution of premating isolation.

Figure 22.6 Reinforcement in phlox. Drummond’s phlox,

Phlox drummondii, is light blue with a pink tinge throughout most of its range (bottom left). However, where it is sympatric with the pointed phlox, D. cuspidata (upper right), it is deep red in color (lower right), making it easier for the butterflies that pollinate them to distinguish between the two species. ©Robin Hopkins

How gene flow may counter speciation Reinforcement is not inevitable, however. When incompletely isolated populations come together, gene flow immediately begins to occur between them. Although hybrids may be inferior, they are not completely inviable or infertile—if they were, the species would already be completely reproductively isolated. When the surviving hybrids reproduce with members of either population, they will serve as a conduit of genetic exchange from one population to the other, and the two populations will tend to lose their genetic distinctiveness. Thus, a race ensues: can complete reproductive isolation evolve before gene flow erases the differences between the populations? The outcome depends on the initial conditions and the particular natural history of the species involved, but many experts consider reinforcement to be the less common outcome.

Learning Outcomes Review 22.2

Natural selection may favor the evolution of increased prezygotic reproductive isolation between sympatric populations when postzygotic isolation initially exists and prezygotic isolation is only partial. This phenomenon is termed reinforcement, and it may lead to the complete reproductive isolation of populations. In contrast, however, genetic exchange between populations may decrease genetic differences among populations, thus preventing speciation from occurring. ■■

How might the initial degree of reproductive isolation affect the probability that reinforcement will occur when two populations come into sympatry?

chapter

22 The Origin of Species 469

22.3 The

Role of Genetic Drift and Natural Selection in Speciation

Learning Outcomes 1. Describe the effects of genetic drift on a population. 2. Explain how genetic drift and natural selection can lead to speciation.

What role does natural selection play in the speciation process? Certainly, the process of reinforcement is driven by natural selection, favoring the evolution of complete reproductive isolation. But reinforcement may not be common. In situations other than reinforcement, does natural selection play a role in the evolution of reproductive isolating mechanisms?

Random changes may cause reproductive isolation As mentioned in chapter 20, populations may diverge as a result of genetic drift. Random change in small populations, founder effects, and population bottlenecks all may lead to changes in traits that cause reproductive isolation. For example, in the Hawaiian Islands, closely related species of Drosophila often differ greatly in their courtship behavior. Colonization of new islands by these fruit flies probably involved a founder effect, in which one or a few flies—perhaps only a single pregnant female—were blown by strong winds to the new island. Changes in courtship behavior between ancestor and descendant populations may be the result of such founder events. Given enough time, any two isolated populations will diverge because of genetic drift (remember that even large populations experience drift, but at a lower rate than in small populations). In some cases, this random divergence may affect traits responsible for reproductive isolation, and speciation may occur.

Adaptation can lead to speciation Although random processes may sometimes be responsible, in many cases natural selection probably plays a role in the speciation process. As populations of a species adapt to different circumstances, they likely accumulate many differences that may lead to reproductive isolation. It is important to realize that in

cases like this, contrary to what occurs during reinforcement, natural selection isn’t acting to favor those individuals that avoid hybridizing with the other species. Rather, increased reproductive isolation evolves as an incidental consequence of the evolution of different adaptations in the different populations. For example, if one population of flies adapts to wet habitats and another to dry areas, then natural selection will favor a variety of corresponding differences in physiological and sensory traits. These differences may produce ecological and behavioral isolation and may cause any hybrids the two populations produce to be poorly adapted to either habitat. Selection on mating behavior might also incidentally lead to the evolution of reproductive isolation. Male Anolis lizards, for example, court females by extending a colorful flap of skin, called a dewlap, located under their throats (­figure  22.7). The ability of one lizard to see the dewlap of another lizard depends not only on the color of the dewlap, but on the environment in which the lizards live. A light-colored dewlap is most effective in reflecting light in a dim forest, whereas dark colors are more apparent in the bright glare of open habitats. As a result, when these lizards occupy new habitats, natural selection favors evolutionary change in dewlap color because males whose dewlaps cannot be seen will not attract many mates. But the lizards also distinguish members of their own species from other species by the color of the dewlap. Adaptive change in mating signals in new environments to enhance visibility to conspecifics could therefore produce reproductive isolation from populations in the ancestral environment; if individuals from the two populations ever came into contact, because of differences in their dewlap colors, they might not recognize each other as members of the same species. Laboratory scientists have conducted experiments on fruit flies and other fast-reproducing organisms in which they isolate populations in different laboratory chambers and measure how much reproductive isolation evolves. These experiments indicate that genetic drift by itself can lead to some degree of reproductive isolation, but in general, reproductive isolation evolves more rapidly when the populations are forced to adapt to different laboratory environments (such as differences in temperature or food type). Although natural selection in the experiment does not directly favor traits because they lead to reproductive isolation, the incidental effect of adaptive divergence is that populations in different environments become reproductively isolated. For this reason, some biologists believe that the term isolating mechanisms is misguided, because it implies that the traits evolved specifically for the purpose of genetically isolating a species, which in most cases—except reinforcement—is probably incorrect.

Figure 22.7 Dewlaps of different species of Caribbean Anolis lizards. Males use their dewlaps in both territorial and courtship

displays. Coexisting species almost always differ in their dewlaps, which are used in species recognition. Darker-colored dewlaps, such as those of the two species on the left, are easier to see in open habitats, whereas lighter-colored dewlaps, like those of the two species on the right, are more visible in shaded environments. Jonathan Losos

b.

a.

c.

Figure 22.8 Populations can become geographically isolated for a variety of reasons. a. Colonization of remote areas by one or a few individuals can establish populations in a distant place. b. Barriers to movement can split an ancestral population into two isolated populations. c. Extinction of intermediate populations can leave the remaining populations isolated from one another.

Learning Outcomes Review 22.3

Genetic drift refers to randomly generated changes in a population’s genetic makeup. Isolated populations will eventually diverge because of genetic drift. Adaptation to different environments may also lead to the reproductive isolation of populations from each other and, in general, is a more potent force producing reproduction isolation. ■■

How is the evolution of reproductive isolation in populations adapting to different environments different from the process of reinforcement?

22.4 The

Geography of Speciation

Learning Outcomes 1. Compare and contrast sympatric and allopatric speciation. 2. Explain the conditions required for sympatric speciation to occur.

Speciation is a two-part process. First, initially identical populations must diverge, and second, reproductive isolation must evolve to maintain these differences. The difficulty with this process, as we have seen, is that the homogenizing effect of gene flow between populations is constantly acting to erase any differences that may arise by genetic drift or natural selection. Gene flow occurs only between populations that are in contact, however, and populations can become geographically isolated for a variety of reasons (figure 22.8). Consequently, evolutionary biologists have long recognized that speciation is much more likely in geographically isolated populations.

Allopatric speciation takes place when populations are geographically isolated Ernst Mayr was the first biologist to demonstrate that geo­­ graphically separated, or allopatric, populations appear much more likely to have evolved substantial differences leading to speciation. Marshalling data from a wide variety of organisms and localities, Mayr made a strong case for allopatric speciation as the primary means of speciation. For example, the little paradise kingfisher varies little throughout its wide range in New Guinea, despite the great variation in the island’s topography and climate. By contrast, isolated populations on nearby islands are strikingly different from one another and from the mainland population (figure 22.9). Thus, geographic isolation seems to have been an important prerequisite for the evolution of differences between populations. Many other examples indicate that speciation can occur under allopatric conditions. Because we would expect isolated populations to diverge over time, by either drift or selection, this result is not surprising. Rather, the more intriguing question becomes: Is geographic isolation required for speciation to occur?

Sympatric speciation occurs without geographic separation For decades, biologists have debated whether one species can split into two at a single locality, without the two new species ever having been geographically separated. Investigators have suggested that this sympatric speciation could occur either instantaneously or over the course of multiple generations. Although most of the hypotheses suggested so far are highly controversial, one type of instantaneous sympatric speciation is known to occur commonly, as the result of polyploidy.

Instantaneous speciation through polyploidy Instantaneous sympatric speciation occurs when an individual is born that is reproductively isolated from all other members of its species. In most cases, a mutation that would cause an individual chapter

22 The Origin of Species 471

Isolated island population of kingfishers

including many of great commercial importance, such as bread wheat, cotton, tobacco, sugarcane, bananas, and potatoes. Speciation by polyploidy is also known to occur in a variety of animals, including insects, fish, and salamanders, although much more rarely than in plants.

Isolated island population of kingfishers

PACIFIC OCEAN

Mainland population of kingfishers

NEW GUINEA

Sympatric speciation by disruptive selection Some investigators believe that sympatric speciation can occur over the course of multiple generations through the process of disruptive selection. As noted in chapter 20, disruptive selection can cause a population to contain individuals exhibiting two different phenotypes. One might think that if selection were strong enough, these two phenotypes would evolve over a number of generations into

Mainland population of kingfishers

Mainland population of kingfishers

Figure 22.9 Phenotypic differentiation in the little paradise kingfisher, Tanysiptera hydrocharis, in New Guinea. Isolated island populations (left) are quite distinctive, showing variation in tail feather structure and length, plumage coloration, and bill size, whereas kingfishers on the mainland (right) show little variation.

to be greatly different from others of its species would have many adverse side effects due to the many pleiotropic effects of most genes (see chapter 12), and the individual likely would not survive. One exception often seen in plants, however, occurs through the process of ­polyploidy, which ­produces individuals that have more than two sets of chromosomes. Polyploid individuals can arise in two ways. In autopolyploidy, all of the chromosomes come from a single species. This might happen, for example, due to an error in cell division that causes a doubling of chromosomes. Such individuals, termed tetra­ ploids because they have four sets of chromosomes, can self-fertilize or mate with other tetraploids, but cannot mate and produce fertile offspring with normal diploids. The reason is that the tetraploid species produce diploid gametes that produce triploid offspring (having three sets of chromosomes) when combined with haploid gametes from normal diploids. Triploids are sterile because the odd number of chromosomes prevents proper pairing during meiosis. A more common type of polyploid speciation is allopolyploidy, which may happen when two species hybridize (figure 22.10). The resulting offspring, having one copy of the chromosomes of each species, is usually infertile because the chromosomes do not pair correctly in meiosis. However, such individuals are often otherwise healthy. Sometimes, the chromosomes of such an individual spontaneously double, as just described for autopolyploidy. Consequently, the resulting tetraploid has two copies of each set of chromosomes and pairing during meiosis is no longer a problem. As a result, such tetraploids are able to interbreed with other similar tetraploids, and a new species has been created. It is estimated that about half of the approximately 260,000 species of plants have a polyploid episode in their history, 472 part IV Evolution

2n=4

2n=6

n=2

n=3

Gametes

Isolated island population of kingfishers

Species 1

Parent Generation

Species 2

F1 Generation: Hybrid Offspring

Doubling of chromosome number

2n=10 Pairing now possible during meiosis

n=5 Viable gametes – sexual reproduction possible with other tetraploid

No doubling of chromosome number

Chromosomes either cannot pair or go through erratic meiosis

No gametes, or sterile gametes – no sexual reproduction possible

Figure 22.10 Allopolyploid speciation. Hybrid offspring

from parents with different numbers of chromosomes often cannot reproduce sexually. Sometimes, the number of chromosomes in such hybrids doubles to produce a tetraploid individual that can undergo meiosis and reproduce with similar tetraploid individuals.

different species. But before the two phenotypes could become different species, they would have to evolve reproductive isolating mechanisms. Initially, the two phenotypes would not be reproductively isolated at all, and genetic exchange between individuals of the two phenotypes would tend to prevent genetic divergence in mating preferences or other isolating mechanisms. As a result, the two phenotypes would be retained as polymorphisms within a single population. For this reason, most biologists consider sympatric speciation of this type to be a rare event. In recent years, however, a number of cases have appeared that are difficult to interpret in any way other than as sympatric speciation. An example occurs on Lord Howe island, a small (16 km2) island, remotely located in the Pacific Ocean 600 km from the nearest large landmass, Australia. The palm genus Howea contains two species, both found on Lord Howe and adapted to living on different types of soil. Given the small size of the island and that the pollen of these trees is dispersed by the wind, little opportunity would have existed for divergence of two populations in isolation from each other. The most reasonable explanation is that the ancestral Howea species colonized the island and subsequently underwent sympatric speciation.

and insects, both of which allowed descendant species to diversify and adapt to many newly available parts of the environment. Adaptive radiation requires both speciation and adaptation to different habitats. A classic model postulates that a species colonizes multiple islands in an archipelago. Speciation subsequently occurs allopatrically, and then the newly arisen species colonize other islands, producing multiple species per island (figure 22.11).

1. An ancestral species flies from mainland to colonize one island.

2. The ancestral species spreads to different islands.

Learning Outcomes Review 22.4

3. Populations on different islands evolve to become different species.

Sympatric speciation occurs without geographic separation, whereas allopatric speciation occurs in geographically isolated populations. Polyploidy and disruptive selection are two ways by which a species may undergo sympatric speciation. ■■

How do polyploidy and disruptive selection differ as ways in which sympatric speciation can occur?

Radiation and Biological Diversity

or

a.

b.

4. Species evolve different adaptations in allopatry.

4. Colonization of islands.

5. Colonization of islands.

5. Species evolve different adaptations to minimize competition with other species (character displacement).

22.5 Adaptive

Learning Outcomes 1. Describe adaptive radiation. 2. List conditions that may lead to adaptive radiation.

One of the most visible manifestations of evolution is the existence of groups of closely related species that have recently evolved from a common ancestor and have adapted to many different parts of the environment. These adaptive radiations are particularly common in situations in which a species occurs in an environment with few other species and many available resources. One example is the creation of new islands through volcanic activity, such as the Hawaiian and Galápagos Islands. Another example is a catastrophic event leading to the extinction of most other species, a phenomenon termed mass extinction that we discuss in section 22.7. Adaptive radiation can also result when a new trait, called a key innovation, evolves within a species, allowing it to use resources or other aspects of the environment that were previously inaccessible. Classic examples of key innovation leading to adaptive radiation are the evolution of lungs in fish and of wings in birds

Figure 22.11 Classic model of adaptive radiation on island archipelagoes. (1) An ancestral species colonizes an island

in an archipelago. Subsequently, the population colonizes other islands (2), after which the populations on the different islands speciate in allopatry (3). Then some of these new species colonize other islands, leading to local communities of two or more species. Adaptive differences can evolve either when species are in allopatry in response to different environmental conditions (a) or as the result of ecological interactions between species (b) by the process of character displacement. chapter

22 The Origin of Species 473

Adaptation to new habitats can occur either during the allopatric phase, as the species respond to different environments on the different islands, or after two species become sympatric. In the latter case, this adaptation may be driven by selective pressures to minimize competition for available resources with other species. In this process, termed character ­displacement, two reproductively isolated but ecologically similar species come into contact. Because the two species use the same resources, natural selection in each species favors those individuals that use resources not used by the other species. Because those individuals will have greater fitness, whatever traits cause the differences in resource use will increase in frequency (assuming that a genetic basis exists for these differences), and, over time, the species will diverge (figure 22.12). One example of character displacement involves three-spined sticklebacks, small fish found in temperate lakes throughout the northern hemisphere. In British Columbia, many lakes were created in the past 12,000 years when the glaciers melted at the end of the last Ice Age. Some of these lakes near the ocean have been invaded by marine populations of the stickleback. These fish tend to occur in SCIENTIFIC THINKING Question: Does competition for resources cause character displacement? Hypothesis: Competition with similar species will cause natural

habitats throughout the lake and evolved to be different from their marine ancestors. So different, in fact, that in the few lakes in which a second colonization occurred, the two stickleback populations had evolved a high degree of reproductive isolation. As a result, natural selection led to the evolution of differences in the two populations to minimize competition for food. One stickleback adapted to using open water by evolving a streamlined body form for greater speed and larger gill structures to more effectively strain out the zooplankton that occur in the open. At the same time, the other population adapted to foraging near the margins and bottom of the lake, evolving a larger and stouter body and altering the shape of the mouth to better feed on large invertebrates from the lake floor (figure 22.13). Similar character displacement occurred in seven doubly colonized lakes. An alternative possibility is that adaptive radiation occurs through repeated instances of sympatric speciation, producing a suite of species adapted to different habitats. As discussed in section 22.4, such scenarios are hotly debated. In this section, we discuss four exemplary cases of adaptive radiation.

?

Inquiry question How would the scenario for adaptive

radiation differ depending on whether speciation is allopatric or sympatric? What is the relationship between character displacement and sympatric speciation?

selection to promote evolutionary divergence. Experiment: Place a species of fish in a pond with another, similar fish species and measure the form of selection. As a control, place a population of the same species in a pond without the second species. Note that the size of food that these fish eat is related to the size of the fish. Result: In the pond with two species, directional selection favors those individuals which have phenotypes most dissimilar from the other species, and thus are most different in resource use. Directional selection does not occur in the control population.

Hawaiian plants and animals exploited a rich, diverse habitat More than 1000 species in the fly genus Drosophila occur on the Hawaiian Islands. New species of Drosophila are still being discovered in Hawaii, although the rapid destruction of the native vegetation is making the search more difficult.

Displacement

Frequency

Frequency

species 1 species 2

Body size

a.

Body size

b.

Interpretation: Would you expect character displacement to occur if resources were unlimited?

Figure 22.12 Character displacement. a. Two species are initially similar and thus overlap greatly in resource use, as might happen if the two species were similar in size (in many species, body size and food size are closely related). Individuals in each species that are most different from the other species (circled) will be favored by natural selection, because they will not have to compete with the other species. For example, the smallest individuals of one species and the largest of the other would not compete with the other species for food and thus would be favored. b. As a result, the species will diverge in resource use and minimize competition between the species. 474 part IV Evolution

Figure 22.13 Stickleback fish adapted to using different habitats. Fish living in open water are more streamlined in body

form, whereas those that occur near the substrate are larger and bulkier, with a mouth better equipped to pick invertebrates off the lake bottom.

a.

b. Figure 22.14 Hawaiian Drosophila. The hundreds of

species that have evolved on the Hawaiian Islands are extremely variable in appearance; note in particular the differences in head shape and body color between these two species. a. Drosophila heteroneura. b. Drosophila grimshawi. Kevin T. Kaneshiro

Aside from their sheer number, Hawaiian Drosophila s­pecies are unusual because of their incredible diversity of ­morphological and behavioral traits (figure 22.14). When their ancestors first reached these islands, they encountered many “empty” habitats that other kinds of insects and other animals occupied elsewhere. As a result, the species have adapted to all manners of fruit fly life and include predators, parasites, and herbivores, as well as species specialized for eating the detritus in leaf litter and the nectar of flowers. The larvae of various species live in rotting stems, fruits, bark, leaves, or roots, or feed on sap. No comparable diversity of Drosophila species is found anywhere else in the world. The great diversity of Hawaiian species is a result of the geological history of these islands. New islands have continually arisen from the sea in this region. As they have done so, they appear to have been invaded successively by the various Drosophila groups present on the older islands. New species thus have evolved as new islands have been colonized. In addition, the Hawaiian Islands are among the most volcanically active islands in the world. Periodic lava flows often have created patches of habitat within an island surrounded by a “sea” of barren rock. These land islands are termed kipukas. Drosophila populations isolated in these kipukas often undergo speciation. In these ways, rampant speciation combined with ecological opportunity has led to an unparalleled diversity of insect life. Similar patterns are shown by many other animal and plant groups. For example, lobeliads are a group of 125 species that have radiated to produce plants ranging in growth form from vines and shrubs to tall trees and that live in habitats as diverse as cliffsides, high-elevation bogs, rainforests, deserts, and coastlines (figure 22.15).

Darwin’s finch species adapted to use different food types The diversity of Darwin’s finches on the Galápagos Islands was first mentioned in chapter 21. Presumably, the ancestor of Darwin’s finches reached these islands before other land birds, and as a result many of the types of habitats that other types of birds use on the mainland were unoccupied. As the new arrivals moved into these vacant ecological niches and adopted new lifestyles, they were subjected to many

a.

b.

c.

d.

Figure 22.15 Hawaiian lobeliads. a. A coastal cliff-dwelling

species, Brighamia rockii, on the cliffs of Molokaì, Hawaiì, b. large tree form of Cyanea hamatiflora from Maui, c. small tree of Cyanea koolauensis from Oahu, and d. bog habitat species Lobelia villosa from Kauai. ©K. R. Wood/NTBG

different sets of selective pressures. Under these circumstances, and aided by the geographic isolation afforded by the many islands of the Galaˊpagos archipelago, the ancestral finches rapidly split into a series of diverse populations, some of which evolved into separate species. These species now occupy many different habitats on the Galápagos Islands, which are comparable to the habitats several distinct groups of birds occupy on the mainland. As illustrated in figure 22.16, the 14 species fall into four groups: 1. Ground and cactus finches. There are six species of Geospiza ground finches. Most of the ground finches feed on seeds. The size of their bills is related to the size of the seeds they eat. Some of the ground finches feed primarily on cactus flowers and fruits, and they have a longer, larger, and more pointed bill than the others. 2. Tree finches. There are five species of insect-eating tree finches. Four species have bills suitable for feeding on insects. The woodpecker finch has a chisel-like beak. This unusual bird carries around a twig or a cactus spine, which it uses to probe for insects in deep crevices. chapter

22 The Origin of Species 475

Ground and Cactus Finches

Geospiza magnirostris

Geospiza scandens

Geospiza conirostris

Geospiza fortis

Geospiza fuliginosa

Vegetarian Tree Finch

Tree Finches

Cactospiza heliobates

Geospiza difficilis

Camarhynchus psittacula

Cactospiza pallida

Camarhynchus pauper

Camarhynchus parvulus

Warbler Finches

Certhidea fusca

Platyspiza crassirostris

Certhidea olivacea

Figure 22.16 An evolutionary tree of Darwin’s finches. This evolutionary tree, derived from the complete genome sequences of Darwin’s finches, indicates that warbler finches are an early offshoot. Ground and tree finches subsequently diverged, and then species within each group specialized to use different resources. Analysis of these genomic data suggested that for several species, such as G. difficilis, what we recognize as a single species that occurs on different islands may instead represent several different species, and these species may not be closely related to each other. Further study is needed to examine this hypothesis. 3. Vegetarian tree finch. The very heavy bill of this species is used to wrench buds from branches. 4. Warbler finches. These unusual birds play the same ecological role in the Gala ́pagos woods that warblers play on the mainland, searching continuously over the leaves and branches for insects. They have slender, warblerlike beaks. Recently, scientists have examined the complete genome sequences of Darwin’s finches to study their evolutionary history. These studies suggest that the deepest branches in the finch evolutionary tree lead to warbler finches, which implies that warbler finches were among the first types to evolve after colonization of the islands. The ground species are closely related to one another, and the same is true for all of the tree finches. Nonetheless, within each group, species differ in beak size and other attributes, as well as in resource use. Field studies, conducted in conjunction with those discussed in chapter 21, demonstrate that ground species compete for resources; the differences between species likely resulted from character displacement as initially similar species diverged to minimize competitive pressures. Recent studies are beginning to uncover the underlying genetic and developmental basis for Darwin’s finch diversification. Work to date has focused on the beaks of the ground finches. Beak variation among species is a result of differential rates of growth in beak height, width, and length. Researchers have identified five 476 part IV Evolution

genes affecting these aspects of beak growth: Bmp4, CaM, TGFβIIr, β-catenin, and Dkk3. In addition, two other genes, ALX1 and HMGA2, have been shown to regulate the activity of other genes that affect beak shape and size. Genetic changes in different combinations of these genes are responsible for the interspecific differences in adult beaks. In addition, one study documented that when character displacement in beak size occurred between two ground finch species, selection acted on HMGA2 to produce the differences in beak size.

Lake Victoria cichlid fishes diversified very rapidly Lake Victoria is an immense, shallow, freshwater sea about the size of Switzerland in the heart of equatorial East Africa. Until recently, the lake was home to an incredibly diverse collection of over 450 species of cichlid fishes.

Geologically recent radiation The cluster of cichlid species appears to have evolved recently and quite rapidly. By sequencing the cytochrome b gene in many of the lake’s fish, scientists have been able to estimate that the first cichlids entered Lake Victoria only 200,000 years ago. Dramatic changes in water level encouraged species formation. As the lake rose, it flooded new areas and opened up new habitats. Many of the species may have originated after the lake

dried down 14,000 years ago, isolating local populations in small lakes until the water level rose again.

Cichlid diversity Cichlids are small, perchlike fishes ranging from 5 to 25 centimeters (cm) in length, and the males come in endless varieties of colors. The ecological and morphological diversity of these fish is remarkable, particularly given the short span of time over which they have evolved. We can gain some sense of the vast range of types by looking at how different species eat. There are mud biters, algae scrapers, leaf chewers, snail crushers, zooplankton eaters, insect eaters, prawn eaters, and fish eaters. Snail shellers pounce on slowcrawling snails and spear their soft parts with long, curved teeth before the snails can retreat into their shells. Scale scrapers rasp slices of scales off other fish. There are even cichlid species that are “pedophages,” eating the young of other cichlids. Cichlid fish have a remarkable key innovation that may have been instrumental in their evolutionary radiation: they carry

a second set of functioning jaws (figure 22.17a). This trait occurs in many other fish, but in cichlids it is greatly enlarged. The ability of these second jaws to manipulate and process food has freed the oral jaws to evolve for other purposes, and the result has been the incredible diversity of ecological roles filled by these fish. In recent years, we have begun to discover the genetic differences underlying the great morphological variety of these fish. For example, extensive genetic analyses have implicated several genes in determining the size and shape of the cichlid jaw (figure 22.17b), including β-catenin, Bmp4, Ptch1, and Lbh. Mutations in these genes in the ancestral cichlid likely allowed individuals to access food in different parts of the habitat, leading to evolutionary divergence and adaptive radiation.

Abrupt extinction in the last several decades Recently, much of the cichlid diversity has disappeared. In the 1950s, the Nile perch, a large commercial fish with a voracious appetite, was introduced to Lake Victoria. Since then, it has spread through the lake, eating its way through the cichlids.

Scale scraper Leaf eater

Second set of jaws Snail eater Fish eater Zooplankton eater Algae scraper Insect eater

a. Labeotropheus fuelleborni Short snout

Maylandia zebra Long snout

b.

Figure 22.17 Cichlid fishes of Lake Victoria. a. These fishes have evolved adaptations to use a variety of different habitats. The enlarged second set of jaws located in the throat of these fish has provided evolutionary flexibility, allowing oral jaws to be modified in many ways. b. A difference in two genes is responsible for a short snout in Labeotropheus fuelleborni and a long snout in Maylandia zebra. (b left) Roberto Nistri/Alamy Stock Photo; (b right) Frank Hecker/Alamy Stock Photo

chapter

22 The Origin of Species 477

By 1990, many of the open-water cichlid species had become extinct, as well as others living in rocky shallow regions. Over 70% of all the named Lake Victoria cichlid species had disappeared, as well as untold numbers of species that had yet to be described. We will revisit the story of Lake Victoria when we discuss conservation biology in chapter 58.

Learning Outcomes Review 22.5

■■

Time

Adaptive radiation occurs when a species diversifies, producing descendant species that are adapted to use many different parts of the environment. Adaptive radiation may occur under conditions of recurrent isolation, which increases the rate at which speciation occurs, and by occupation of areas with few competitors and many types of available resources, such as on volcanic islands. The evolution of a key innovation may also allow adaptation to parts of the environment that previously couldn’t be utilized. In contrast to the archipelago model, how might an adaptive radiation proceed in a case of sympatric speciation by disruptive selection?

22.6 The

Pace of Evolution

Learning Outcomes 1. Define stasis and compare it to gradual evolutionary change. 2. Explain the components of the punctuated equilibrium hypothesis.

We have discussed the manner in which speciation may occur, but we haven’t yet considered the relationship between speciation and the evolutionary change that occurs within a species. Two hypotheses, gradualism and punctuated equilibrium, have been advanced to explain the relationship.

Gradualism is the accumulation of small changes For more than a century after the publication of On the Origin of Species, the standard view was that evolution occurred very slowly. Such change would be nearly imperceptible from generation to generation, but would accumulate such that, over the course of thousands and millions of years, major changes­could occur. This view is termed gradualism (figure 22.18a).

Punctuated equilibrium is long periods of stasis followed by relatively rapid change An alternative possibility is that species experience long periods of little or no evolutionary change (termed stasis), punctuated by bursts of evolutionary change occurring over geologically short 478 part IV Evolution

a. Gradualism

b. Punctuated equilibrium

Figure 22.18 Two views of the pace of macroevolution. 

a. Gradualism suggests that evolutionary change occurs slowly through time and is not linked to speciation, whereas b. punctuated equilibrium requires that phenotypic change occurs in bursts associated with speciation, separated by long periods of little or no change.

time intervals. This phenomenon is termed punctuated ­equilibrium (­figure  22.18b); some have argued that these periods of rapid change occurred only during the speciation process. We have seen in chapters 20 and 21 that when natural selection is strong, evolutionary change can occur rapidly, so the “punctuated” part of the theory is not controversial. A more difficult question involves the long periods of stasis (the equilibrium): Why would species exist for thousands, or even millions, of years without changing? Although a number of possible reasons have been suggested, most researchers now believe that a combination of stabilizing and oscillating selection is responsible for stasis. If the environment does not change over long periods of time, or if environmental changes oscillate back and forth, then stasis may occur for long periods. One factor that may enhance this stasis is the ability of species to shift their ranges; for example, during the ice ages, when the global climate cooled, the geographic ranges of many species shifted southward, so that the species continued to experience similar environmental conditions.

?

Inquiry question Why would changes in geographic ranges promote evolutionary stasis?

Evolution may include both types of change

22.7 Speciation

and Extinction Through Time

The fossil record shows that some well-documented groups, such as African mammals, clearly have evolved gradually, not in spurts. Other groups, such as marine bryozoa, seem to show the irregular pattern of evolutionary change predicted by the punctuated equilibrium model. It appears, in fact, that gradualism and punctuated equilibrium are two ends of a continuum. Although some groups have evolved solely in a gradual manner and others only in a punctuated mode, many other groups show evidence of both gradual and punctuated episodes at different times in their evolutionary history. The idea that speciation is necessarily linked to phenotypic change has not been supported, however. On one hand, it is now clear that speciation can occur without substantial phenotypic change. For example, many closely related salamander species are nearly indistinguishable. On the other hand, it is also clear that phenotypic change can occur within species in the absence of speciation.

Learning Outcomes 1. Describe the pattern of species diversity through time. 2. Define mass extinction and identify when major mass extinctions have occurred.

Biological diversity has increased vastly over the last 600 million years, but the trend has been far from consistent. After a rapid rise, diversity reached a plateau for about 200 million years, but since then has risen steadily. Because changes in the number of species reflect the rate of origin of new species relative to the rate at which existing species disappear, this long-term trend reveals that speciation has, in general, surpassed extinction. Nonetheless, speciation has not always outpaced extinction. In particular, interspersed in the long-term increase in species diversity have been a number of sharp declines, termed mass extinctions.

Learning Outcomes Review 22.6

Gradualism is the accumulation of almost imperceptible changes that eventually results in major differences. Punctuated equilibrium proposes that long periods of stasis are interrupted (punctuated) by periods of rapid change. Evidence for both gradualism and punctuated equilibrium has been found in different groups. Stasis refers to a period in which little or no evolutionary change occurs. Stasis may result from stabilizing or oscillating selection. ■■

Five mass extinctions have occurred in the distant past Five major mass extinctions have been identified, the most severe one occurring at the end of the Permian period, approximately 250 million years ago (mya) (figure 22.19). At that time, more than half of all plant and animal families and as many as 96% of all species may have perished.

Could evolutionary change be punctuated in time (that is, rapid and episodic), but not linked to speciation?

Figure 22.19 

Biodiversity through time. The taxonomic diversity

1000

Number of families

800

600 Cretaceous 400 Devonian Ordovician

200

Permian Triassic

0

600

500

400

300

200

100

0

of families of marine animals has increased through time, although occasional dips have occurred. The fossil record is most complete for marine organisms because they are more readily fossilized than terrestrial species. Families are shown, rather than species, because many species are known from only one specimen, thus introducing error into counts of the number of species present at a particular point in time. Arrows indicate the five major mass extinction events.

Millions of years ago

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22 The Origin of Species 479

The most famous and well-studied extinction, although not as drastic, occurred at the end of the Cretaceous period (66 mya), at which time the dinosaurs (except for birds, which are one type of dinosaur—see chapter 34) and a variety of other organisms went extinct. Recent findings have supported the hypothesis that this extinction event was triggered when a large asteroid slammed into Earth, perhaps causing global forest fires and obscuring the Sun for months by throwing particles into the air. The cause of other mass extinction events is less certain. Some scientists suggest that asteroids may have played a role in at least some of the other mass extinction events; other hypotheses implicate global climate change, massive volcanic eruptions, and other causes. One important result of mass extinctions is that not all groups of organisms are affected equally. For example, in the extinction at the end of the Cretaceous, not only most dinosaurs, but also marine and flying reptiles and ammonites (a type of mollusk) went extinct. Marsupials, flowering plants, birds, and some forms of plankton were greatly reduced in diversity. In contrast, turtles, crocodilians, and amphibians seemed to have been unscathed. Why some groups were harder hit than others is not clear, but one hypothesis suggests that survivors were those animals that could shelter underground or in water, and that either could scavenge or required little food in the cool temperatures that resulted from the blockage of sunlight. A consequence of mass extinctions is that previously dominant groups may perish, thus changing the course of evolution. This is certainly true of the Cretaceous extinction. During the Cretaceous period, placental mammals were a minor group composed of species that were mostly no larger than a house cat. When the dinosaurs, which had dominated the world for more than 100 million years, disappeared at the end of this period, the placental mammals underwent a significant adaptive radiation. It is humbling to think that humans might never have arisen had that asteroid not struck Earth 65 mya. As the world around us illustrates today, species diversity does rebound after mass extinctions, but this recovery is not rapid. Examination of the fossil record indicates that rates of speciation

do not immediately increase after an extinction pulse, but rather take about 10 million years to reach their maximum. The cause of this delay is not clear, but it may result because it takes time for ecosystems to recover and for the processes of speciation and adaptive diversification to begin. Consequently, species diversity may require 10 million years, or even much longer, to attain its previous level.

A sixth mass extinction is under way The number of species in the world in recent times is greater than it has ever been. Unfortunately, that number is decreasing at an alarming rate due to human activities (see chapter 58). Some estimate that as much as one-fourth of all species will become extinct in the near future, a rate of extinction not seen on Earth since the Cretaceous mass extinction. Moreover, the rebound in species diversity may be even slower than following previous mass extinction events because, instead of the ecologically impoverished but energy-rich environment that existed after previous mass extinction events, a large proportion of the world’s resources will be taken up already by human activities, leaving few resources available for adaptive radiation. The current species extinction crisis is discussed in greater detail in chapter 58.

Learning Outcomes Review 22.7

The number of species has increased through time, although not at a constant rate. Five major extinction events have substantially, although briefly, reduced the number of species. Diversity rebounds, but the recovery is not rapid, and the groups making up that diversity are not the same as those that existed before the extinction event. Unfortunately, humans are currently causing a sixth mass extinction event. ■■

In what ways is the current mass extinction event different from those that have occurred in the past?

Chapter Review JohnMernick/iStock/Getty Images

22.1 The Nature of Species and the Biological

Species Concept

Sympatric species inhabit the same locale but remain distinct. Most sympatric species are readily distinguishable phenotypically and ecologically. Others usually can be identified by careful study.

480 part IV Evolution

Populations of a species exhibit geographic variation. Populations that differ greatly in phenotype or ecologically are usually connected by geographically intermediate populations. The biological species concept focuses on the ability to exchange genes. In the biological species concept, species are defined as those populations that interbreed, or have the potential to do so, and produce

fertile offspring. Reproductive isolating mechanisms prevent genetic exchange between species.

one species to divide into two, but scientists debate how commonly it occurs.

Prezygotic isolating mechanisms prevent the formation of a zygote. Prezygotic isolating mechanisms prevent a viable zygote from being created. These mechanisms include ecological, behavioral, temporal, and mechanical isolation, as well as failure of gametes to unite to form a zygote.

22.5 Adaptive Radiation and Biological Diversity

Postzygotic isolating mechanisms prevent normal development into reproducing adults. Postzygotic isolating mechanisms prevent a zygote from developing into a viable and fertile individual. The biological species concept does not explain all observations. An alternative explanation for the existence of distinct species focuses on the role of natural selection and differences among species in their ecological requirements. In reality, no one species concept can explain all the diversity of life.

22.2 Natural Selection and Reproductive Isolation Selection may reinforce isolating mechanisms. Populations may evolve complete reproductive isolation in allopatry. If populations that have evolved only partial reproductive isolation come into contact, natural selection can lead to increased reproductive isolation, a process termed “reinforcement.” Alternatively, genetic exchange between the populations can lead to homogenization and thus prevent speciation from occurring.

22.3 The Role of Genetic Drift and Natural Selection in

Speciation

Random changes may cause reproductive isolation. In small populations, genetic drift may cause populations to diverge; the differences that evolve may cause the populations to become reproductively isolated. Adaptation can lead to speciation. Adaptation to different situations or environments may incidentally lead to reproductive isolation. In contrast to reinforcement, these differences are not directly favored by natural selection because they prevent hybridization.

22.4 The Geography of Speciation (figure 22.9) Allopatric speciation takes place when populations are geographically isolated. Allopatric, or geographically isolated, populations may evolve into separate species because no gene flow occurs between them. Most speciation probably occurs in allopatry. Sympatric speciation occurs without geographic separation. Sympatric speciation can occur in two ways. One is polyploidy, which instantly creates a new species. Disruptive selection also can cause

Adaptive radiation occurs when a species finds itself in a new or suddenly changed environment with many resources and few competing species (figure 22.11). The evolution of a new trait that allows individuals to use previously inaccessible parts of the environment, termed a “key innovation,” may also trigger an adaptive radiation. Hawaiian plants and animals exploited a rich, diverse habitat. At least 1000 species of Hawaiian Drosophila have been identified. Several groups of plants have also diversified to occupy many habitats. Darwin’s finch species adapted to use different food types. Fourteen species in four genera have evolved to exploit four different habitats based on type of food. Lake Victoria cichlid fishes diversified very rapidly. Cichlids underwent rapid radiation to form more than 450 species, although today more than 70% of these species are now extinct.

22.6 The Pace of Evolution Gradualism is the accumulation of small changes. Historically, scientists took the view that speciation occurred gradually through very small cumulative changes. Punctuated equilibrium is long periods of stasis followed by relatively rapid change. The punctuated equilibrium hypothesis contends that not only is change rapid and episodic, but also it is only associated with the speciation process. Evolution may include both types of change. Scientists generally agree that evolutionary change occurs on a continuum, with gradualism and punctuated change being the extremes.

22.7 Speciation and Extinction Through Time In general, the number of species has increased through time (figure 22.19). Five mass extinctions have occurred in the distant past. Mass extinctions have led to dramatic decreases in species diversity followed by recovery that takes millions of years. Five such mass extinctions have occurred in the distant past due to asteroids hitting the Earth and global climate change, among other events. A sixth mass extinction is under way. Humans are currently causing a sixth mass extinction.

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22 The Origin of Species 481

Visual Summary Genetic drift

may evolve due to

Species

defined by

Biological species concept

requires

limitations lead to

Natural selection

can be shaped by

can occur by

Altenative concepts

Prezygotic isolation

such as Ecological species concept Allopatry occurs in

Reproductive isolation

affects

Geography

Sympatry

affects

Postzygotic isolation leads to

Speciation

may affect

Genetic drift

and extinction produce varies in

Pace of evolution

occurs rapidly

occurs slowly

Punctuated equilibrium

Gradualism

Evolutionary diversification

decreases during

Mass extinctions

can produce Adaptive radiation

Review Questions JohnMernick/iStock/Getty Images

U N D E R S TA N D 1. Prezygotic isolating mechanisms include all of the following except a. hybrid sterility. b. courtship rituals.

c. habitat separation. d. seasonal reproduction.

2. Reproductive isolation is a. b. c. d.

a result of individuals not mating with each other. a specific type of postzygotic isolating mechanism. required by the biological species concept. None of the choices is correct.

3. Problems with the biological species concept include the fact that a. b.

many species reproduce asexually. postzygotic isolating mechanisms decrease hybrid viability.

482 part IV Evolution

c. d.

prezygotic isolating mechanisms are extremely rare. All of the choices are correct.

4. Allopatric speciation a. b. c. d.

is less common than sympatric speciation. involves geographic isolation of some kind. is the only kind of speciation that occurs in plants. requires polyploidy.

5. Gradualism and punctuated equilibrium are a. b. c. d.

two ends of the continuum of the rate of evolutionary change over time. mutually exclusive views about how all evolutionary change takes place. mechanisms of reproductive isolation. None of the choices is correct.

6. Prezygotic isolation a. b. c. d.

always involves mechanisms that prevent interbreeding of members of different species. includes the death of a zygote shortly after fertilization. occurs only in plants. becomes stronger as the result of reinforcement.

7. Speciation by allopolyploidy a. b. c. d.

takes a long time. is common in birds. leads to reduced numbers of chromosomes. occurs after hybridization between two species.

8. Adaptive radiation a. b. c. d.

is the result of enriched uranium used in power plants. is the evolution of closely related species adapted to use different parts of the environment. results from genetic drift. is the outcome of stabilizing selection favoring the maintenance of adaptive traits.

9. Leopard frogs from different geographic populations of the Rana pipiens complex a. b. c. d.

are members of a single species because they look very similar to one another. are different species shown to have pre- and postzygotic isolating mechanisms. frequently interbreed to produce viable hybrids. are genetically identical due to effective reproductive isolation.

10. Character displacement a. b. c. d.

arises through competition and natural selection, favoring divergence in resource use. arises through competition and natural selection, favoring convergence in resource use. does not promote speciation. reduced speciation rates in Galápagos finches.

11. During the history of life on Earth a. b. c. d.

there have been major extinction events. species diversity has steadily increased. species diversity has stayed relatively constant. extinction rates have been completely offset by speciation rates.

A P P LY 1. If reinforcement is weak and hybrids are not completely infertile, a. b.

genetic divergence between populations may be overcome by gene flow. speciation will occur 100% of the time.

c. d.

gene flow between populations will be impossible. the speciation will be more likely than if hybrids were completely infertile.

2. Natural selection can a. b. c. d.

enhance the probability of speciation. enhance reproductive isolation. act against hybrid survival and reproduction. All of the choices are correct.

3. Hybridization between incompletely isolated populations a. b. c. d.

always leads to reinforcement due to the inferiority of hybrids. can serve as a mechanism for preserving gene flow between populations, thus preventing speciation. occurs only in plants. never affects rates of speciation.

4. Natural selection can lead to speciation a. b. c. d.

by causing small populations to diverge more than large populations. because the evolutionary changes that two populations acquire while adapting to different habitats may have the effect of making them reproductively isolated. by favoring the same evolutionary change in multiple populations. by favoring intermediate phenotypes.

SYNTHESIZE 1. Natural selection can lead to the evolution of prezygotic isolating mechanisms, but not that of postzygotic isolating mechanisms. Explain. 2. What role does natural selection play in the origin of species? How might your answer depend on how you define what a species is? 3. Refer to figure 22.6. In Texas, Drummond’s phlox is a dark shade of red where it occurs with the pale blue pointed phlox. Elsewhere, where it occurs by itself, it is a pale pinkish-blue. In this case, there is no competition for ecological resources as in other cases of character divergence discussed. Why has the color of Drummond’s phlox evolved when it is sympatric with pointed phlox? 4. Refer to figure 22.16. Geospiza fuliginosa and Geospiza fortis are found in sympatry on at least one island in the Galápagos and in allopatry on several islands in the same archipelago. Compare your expectations about degree of morphological similarity of the two species in these two contexts, given the hypothesis that competition for food played a large role in the adaptive radiation of this group. Would your expectations be the same for a pair of finch species that are not as closely related? Explain.

chapter

22 The Origin of Species 483

23 CHAPTER

Systematics, Phylogenies, and Comparative Biology Chapter Contents 23.1

Systematics

23.2 Cladistics 23.3 Systematics and Classification 23.4 Phylogenetics and Comparative Biology 23.5 Phylogenetics and Disease Evolution

Imagemore Co, Ltd./Imagemore/Getty Images

Introduction

Visual Outline Evolutionary relationships

depicted by

inferred by

Cladistics Lamprey

Shark

Salamander Lizard

Tiger

Gorilla Human

Phylogenies used to infer

Evolution

ordered into

Classifications

Domain Eukarya Kingdom Animalia Phylum Chordata Subphylum Vertebrata Class Mammalia Order Rodentia Family Sciuridae Genus Sciurus Species Sciurus carolinensis

Sciurus carolinensis

All organisms share many biological characteristics. They are composed of one or more cells, carry out metabolism and transfer energy with ATP, and encode hereditary information in DNA. Yet, there is also a tremendous diversity of life, ranging from bacteria and amoebas to blue whales and sequoia trees. The photograph above shows variation in one particularly diverse group of organisms, the beetles. For generations, biologists have tried to group organisms based on shared characteristics. The most meaningful groupings are based on the study of evolutionary relationships among organisms. New methods for constructing evolutionary trees and a sea of molecular sequence data are leading to improved evolutionary hypotheses to explain life’s diversification.

by new data, leading to the formation of better, more accurate scientific ideas. The study of evolutionary relationships is called systematics. By looking at the similarities and differences between species, systematists can construct an evolutionary tree, or phylogeny, which represents a hypothesis about patterns of relationship among species.

23.1 Systematics Learning Outcomes 1. Understand what a phylogeny represents. 2. Explain why phenotypic similarity does not necessarily indicate a close evolutionary relationship.

Branching diagrams depict evolutionary relationships Darwin envisioned that all species were descended from a single common ancestor, and that the history of life could be depicted as a branching tree (figure 23.1). In Darwin’s view, the twigs of the tree represent existing species. As one works down the tree, the joining of twigs and branches reflects the pattern of common ancestry back in time to the single common ancestor of all life. The process of descent with modification from common ancestry results in all species being related in this branching, hierarchical fashion, and their evolutionary history can be depicted using branching diagrams referred to as phylogenetic trees. Figure 23.1b shows how evolutionary relationships are depicted with a branching diagram. Humans and chimpanzees are descended from a common ancestor and are each other’s closest living relative (the position of this common ancestor is indicated by the node labeled 1). Humans, chimps, and gorillas share an

One of the great challenges of modern science is to understand the history of ancestor–descendant relationships that unites all forms of life on Earth, from the earliest single-celled organisms to the complex organisms we see around us today. If the fossil record were perfect, we could trace the evolutionary history of species and examine how each arose and proliferated. However, as discussed in chapter 21, the fossil record is far from complete, answering many questions about life’s diversification, but leaving many others unsettled. Consequently, scientists must rely on other types of evidence to establish the best hypothesis of evolutionary relationships. Bear in mind that the outcomes of such studies are hypotheses, and as such, they require further testing. All hypotheses may be disproved

Variations of a Cladogram Gibbon

Human

Chimp

Gorilla

Orangutan

Gibbon Orangutan

Gorilla

Human Chimp 1

1 2

2 Chimp

3

3

1

Version 1

2 3

Version 2

Human Gorilla Orangutan Gibbon Version 3

a.

b.

Figure 23.1 Phylogenies depict evolutionary relationships. a. A drawing from one of Darwin’s notebooks, written in 1837 as he

developed his ideas that led to On the Origin of Species. Darwin viewed life as a branching process akin to a tree, with species on the twigs, and evolutionary change represented by the branching pattern displayed by a tree as it grows. b. An example of a phylogeny. Note that these three versions convey the same information despite the differences in arrangement of species and orientation. Humans and chimpanzees are more closely related to each other than they are to any other living species. This is apparent because they share a common ancestor (the node labeled 1) that was not an ancestor of other species. Similarly, humans, chimpanzees, and gorillas are more closely related to one another than any of them is to orangutans because they share a common ancestor (node 2) that was not ancestral to orangutans. Node 3 represents the common ancestor of all apes. At each node, the two descendants can be rotated without changing the meaning. For example, one difference between versions 1 and 2 is that the descendants of node 2 have been rotated so that gorilla branches to the right in version 1 and to the left in version 2. However, this does not affect the interpretation that humans and chimps are more closely related to each other than either species is to gorillas. (a) Letz/SIPA/Newscom chapter

23 Systematics, Phylogenies, and Comparative Biology 485

older common ancestor (node 2), and all apes share a more distant common ancestor (node 3). One key to interpreting a phylogeny is to look at how recently species share a common ancestor, rather than looking at the arrangement of species across the top of the tree. If you compare the three versions of the phylogeny of figure 23.1b, you can see that the relationships are the same: regardless of where they are positioned, chimpanzees and humans are still more closely related to each other than to any other species. Moreover, even though humans are placed next to gibbons in version 1 of figure 23.1b, the pattern of relationships still indicates that humans are more closely related (that is, share a more recent common ancestor) with gorillas and orangutans than with gibbons. Phylogenies are also sometimes displayed on their side, rather than upright (figure 23.1b, version 3), but this arrangement also does not affect its interpretation.

Similarity may not accurately predict evolutionary relationships We might expect that the greater the time since two species diverged from a common ancestor, the more different they would be. Early systematists relied on this reasoning and constructed phylogenies based on overall similarity. If, in fact, s­ pecies evolved at a constant rate, then the amount of divergence between two species would be a function of how long they had been diverging, and thus phylogenies based on degree of similarity would be accurate. As a result, we might think that chimps and gorillas are more closely related to each other than either is to humans. But as chapter 22 revealed, evolution can occur very rapidly at some times and very slowly at others. Species invading new habitats are likely to experience new selective pressures and may change greatly; those staying in the same habitats as their ancestors may change only a little. For this reason, rates of change may vary markedly among species. Consequently, some will diverge more than others, and as a result, similarity is not necessarily a good predictor of how long it has been since two species shared a common ancestor. A second fundamental problem exists as well: evolution is not always divergent. In chapter 21, we discussed convergent evolution, in which two species independently evolve the same features. Often, species evolve convergently because they use similar environments, in which similar adaptations are favored. As a result, two species that are not closely related may end up more similar to each other than they are to their close relatives. Evolutionary reversal, the process in which a species re-evolves the characteristics of an ancestral species, also has this effect.

Learning Outcomes Review 23.1

Systematics is the study of evolutionary relationships. Phylogenies, or phylogenetic trees, are graphic representations of relationships among species. Similarity of organisms alone does not necessarily correlate with their relatedness because evolutionary change is not constant in rate and direction. ■■

Why might a species be most phenotypically similar to a species that is not its closest evolutionary relative?

486 part IV Evolution

23.2 Cladistics Learning Outcomes 1. Describe the difference between ancestral and derived similarities. 2. Explain why only shared derived characters indicate close evolutionary relationship. 3. Demonstrate how a cladogram is constructed.

Because phenotypic similarity may be misleading, most systematists no longer construct their phylogenetic hypotheses solely on this basis. Rather, they distinguish similarity among species that is inherited from the most recent common ancestor of an entire group, which is called ancestral, from similarity that arose more recently—that is, is not inherited from the most recent common ancestor of the group—and is shared only by a subset of the species; this similarity is called derived. In this approach, termed cladistics, only shared ­derived characters are considered informative in determining evolutionary relationships.

The cladistic method requires that character variation be identified as ancestral or derived To employ the method of cladistics, systematists first gather data on a number of characters for all the species in the analysis. Characters can be any aspect of the phenotype, including morphology, physiology, behavior, and DNA. As chapters 18 and 24 show, the revolution in genomics is now providing a vast body of data revolutionizing our ability to identify and study character variation. To be useful, the characters should exist in recognizable character states. For example, consider the character “tail” in vertebrates. This character has two states: presence in most vertebrates and absence in humans, apes, and some other groups, such as frogs. A cladistic analysis begins by determining the states for a number of characters for each taxon (taxa are species or higherlevel groups, such as genera or families) in the analysis. In the example in figure 23.2, six characters are used for seven taxa. The first character, presence of jaws, is scored as absent in lamprey (denoted by 0) and present in the other species (denoted with a 1). Another character, hair, is absent in all of the taxa except the three mammal species.

Examples of ancestral versus derived characters The presence of hair is a shared derived feature of mammals (figure 23.2); in contrast, the presence of lungs in mammals is an ancestral feature because it is also present in amphibians and reptiles (represented by a salamander and a lizard) and therefore presumably evolved prior to the common ancestor of mammals (figure 23.2). The presence of lungs, therefore, does not tell us that mammal species are all more closely related to one another than to reptiles or amphibians, but the shared, derived feature of hair suggests that all mammal species share a common ancestor that existed more recently than the common ancestor of mammals, amphibians, and reptiles.

Jaws

Lungs

Amniotic Membrane

Hair

No Tail

Bipedal

Lamprey

0

0

0

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0

0

Shark

1

0

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0

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0

Salamander

1

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0

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Lizard

1

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Gorilla

1

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1

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Traits: Organism

Lamprey

Shark

Salamander

Lizard

Tiger

Gorilla

Human

Bipedal Tail loss Hair Amniotic membrane Lungs Jaws

a.

b.

Figure 23.2 A cladogram. a. Morphological data for a group of seven vertebrates are tabulated. A “1” indicates possession of the derived

character state, and a “0” indicates possession of the ancestral character state (note that the derived state for character “no tail” is the absence of a tail; for all other traits, absence of the trait is the ancestral character state). b. A tree, or cladogram, diagrams the relationships among the organisms based on the presence of derived characters. The derived characters between the cladogram branch points are shared by all organisms above the branch points and are not present in any below them. The outgroup (in this case, the lamprey) does not possess any of the derived characters.

To return to the question concerning the relationships of humans, chimps, and gorillas, a number of morphological and DNA characters exist that are derived and shared by chimps and humans, but not by gorillas or other great apes. These characters suggest that chimps and humans diverged from a common ancestor (figure 23.1b, node 1) that existed more recently than the common ancestor of gorillas, chimps, and humans (node 2).

Determination of ancestral versus derived Once the data are assembled, the first step in a cladistic analysis is to polarize the characters—that is, to determine whether particular character states are ancestral or derived. To polarize the character “tail,” for example, systematists must determine which state— presence or absence—was exhibited by the most recent common ancestor of this group. Usually, the fossils available do not include the most recent common ancestor—or at least we cannot be confident that they do. As a result, the method of outgroup comparison is used to assign character polarity. To use this method, a species or group of species that is closely related to, but not a member of, the group under study is designated as the outgroup. When the group under study exhibits multiple character states, and one of those states is exhibited by the outgroup, then that state is considered to be ancestral and other states are considered to be derived. However, outgroup species also evolve from their ­ancestors, so the outgroup species will not always exhibit the ancestral condition. For this reason, polarity assignments are most reliable when the same character state is exhibited by several different outgroups. In the preceding example, a tail is generally present in the nearest outgroups of vertebrates. Consequently, the presence of a tail in vertebrates is considered ancestral, and its absence in some species is considered derived.

Construction of a cladogram Once all characters have been polarized, systematists use this information to construct a cladogram, which depicts a hypothesis of evolutionary relationships (a cladogram is the type of phylogenetic tree produced by cladistic analysis). Species that share a common ancestor, as indicated by the possession of shared derived characters, are said to belong to a clade. Clades are thus evolutionary units and refer to a common ancestor and all of its descendants. A derived character shared by clade members is called a synapomorphy of that clade. Figure 23.2b illustrates that a simple cladogram is a nested set of clades, each characterized by its own synapomorphies. For example, amniotes are a clade for which the evolution of an amniotic membrane is a synapomorphy. Within that clade, mammals are a clade, with hair as a synapomorphy, and so on. Ancestral states are called plesiomorphies, and shared ancestral states are called symplesiomorphies. In contrast to synapomorphies, symplesiomorphies are not informative about phylogenetic relationships. Let’s return to the character state “presence of a tail,” which is exhibited by lampreys, sharks, salamanders, lizards, and tigers. Does this mean that tigers are more closely related to—and shared a more recent common ancestor with—lizards and sharks than to apes and humans, their fellow mammals? The answer, of course, is no: because symplesiomorphies reflect character states inherited from a distant ancestor, they do not imply that species exhibiting that state are closely related.

Homoplasy complicates cladistic analysis In real-world cases, phylogenetic studies are rarely as simple as the examples we have shown so far. The reason is that in some cases, the same character has evolved independently in several species. chapter

23 Systematics, Phylogenies, and Comparative Biology 487

Salamander

Frog

Lizard

Tiger

Gorilla

Human

Salamander

Lizard

Tiger

Frog

Hair loss Amniotic membrane loss

Tail loss Tail loss Hair

Human

Tail loss Hair

Amniotic membrane

a.

Gorilla

Amniotic membrane

b.

Figure 23.3 Parsimony and homoplasy. a. The placement of frogs as closely related to salamanders requires that tail loss evolved twice,

an example of homoplasy. b. If frogs are closely related to gorillas and humans, then tail loss had to evolve only once. However, this arrangement would require two additional evolutionary changes: frogs would have to have lost the amniotic membrane and hair (alternatively, hair could have evolved independently in tigers and the clade of humans and gorillas; this interpretation would require two evolutionary changes, hair evolving twice, just like the interpretation shown in the figure, in which hair evolved only once, but then was lost in frogs). Based on the principle of parsimony, the cladogram that requires the fewest number of evolutionary changes is favored; in this case the cladogram in (a) requires four changes, whereas that in (b) requires five, so (a) is considered the preferred hypothesis of evolutionary relationships.

Data analysis Construct a data matrix like the one in figure 23.2, showing the character states for all six species for the traits hair, amniotic membrane, and tail. These characters would be categorized as shared derived characters, but they would be false signals of a close evolutionary relationship. In addition, derived characters may sometimes be lost as species within a clade re-evolve to the ancestral state. Homoplasy refers to a shared derived character state that has not been inherited from a common ancestor exhibiting that character state. Homoplasy can result from convergent evolution or from evolutionary reversal. For example, adult frogs do not have a tail. Thus, absence of a tail is a synapomorphy that unites not only gorillas and humans, but also frogs. However, frogs are not closely related to gorillas and humans; they have neither an amniotic membrane nor hair, both of which are synapomorphies for clades that contain gorillas and humans. In cases such as this, when there are conflicts among the characters, systematists rely on the principle of parsimony, which favors the hypothesis that requires the fewest assumptions. As a result, the phylogeny that requires the fewest evolutionary events is considered the best hypothesis of phylogenetic relationships (figure 23.3). In the example just stated, therefore, grouping frogs with salamanders is favored because it requires only one instance of homoplasy (the multiple origins of taillessness), whereas a phylogeny in which frogs were most closely related to humans and gorillas would require two homoplastic evolutionary events (the loss of both amniotic membranes and hair in frogs). The examples presented so far have all involved morphological characters, but systematists increasingly use DNA sequence data to construct phylogenies because of the large number of characters that can be obtained through sequencing. Cladistics analyzes 488 part IV Evolution

sequence data in the same manner as any other type of data: character states are polarized by reference to the sequence of an outgroup, and a cladogram is constructed that minimizes the amount of character evolution required ­(figure 23.4).

Other phylogenetic methods work better than cladistics in some situations If characters evolve from one state to another at a slow rate compared with the frequency of speciation events, then the principle of parsimony works well in reconstructing evolutionary relationships. In this situation, the principle’s underlying assumption—that shared derived similarity is indicative of recent common ancestry—is usually correct. In recent years, however, systematists have realized that some characters evolve so rapidly that the principle of parsimony may be misleading.

Rapid rates of evolutionary change and homoplasy Of particular interest is the rate at which some parts of the genome evolve. As discussed in chapter 18, some stretches of DNA do not appear to have any function. As a result, mutations that occur in these parts of the DNA are not eliminated by natural selection, and thus the rate of evolution of new character states can be quite high in these regions as a result of genetic drift. Moreover, because only four character states are possible for any nucleotide base (A, C, G, or T), there is a high probability that two species will independently evolve the same derived character state at any particular base position. If such homoplasy dominates the character data set, then the assumptions of the principle of

Outgroup

DNA Sequence Site

1

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Species A

G

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A

Species C Species D Outgroup

G A

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T T

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G

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Species B

Species D

8:T

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4:T

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8:T

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10:T

T

A

T

C

C

G

T

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G

T G 2:T

A

Species A

6:C

G

9:A

G

C

T

Species C

C 1:A

G

5:C

A

Homoplastic evolutionary changes Nonhomoplastic evolutionary changes

Figure 23.4 Cladistic analysis of DNA sequence data. Sequence data are analyzed just like any other data. The most parsimonious

interpretation of the DNA sequence data requires nine evolutionary changes. Each of these changes is indicated on the phylogeny. Change in site 8 is homoplastic: species A and B independently evolved from thymine to cytosine at that site. Nonhomoplastic changes are those in which all species that possess the trait derived it from the same common ancestor. Such traits are referred to as homologous.

parsimony are violated: similarity of species in this situation would more likely result from homoplasy than from inheritance from a common ancestor. As a result, under these circumstances, phylogenies inferred using the cladistic method are likely to be inaccurate.

?

Inquiry question Why do high rates of evolutionary change and a limited number of character states cause problems for parsimony analyses?

Statistical approaches Because evolution can sometimes proceed rapidly, systematists in recent years have been exploring other methods based on statistical approaches, such as maximum likelihood, to infer phylogenies. These methods start with an assumption about the rate at which characters evolve and then fit the data to these models to derive the phylogeny that best accords (that is, “is maximally likely”) with these assumptions. One advantage of these methods is that different assumptions of rate of evolution can be used for different characters. If some DNA characters evolve more slowly than others—for example, because they are constrained by natural selection—then the methods can employ different models of evolution for the different characters. This approach is more effective than parsimony in dealing with homoplasy when rates of evolutionary changes are high.

The molecular clock In general, cladograms such as the one in figure 23.2 indicate only the order of evolutionary branching events; they do not contain information about the timing of these events. In some cases, however, branching events can be timed, either by reference to fossils, or by making assumptions about the rate at which characters change. One widely used but controversial method is the molecular clock, which states that the rate of evolution of a molecule is constant through time. In this model, divergence in DNA can be used to calculate the times at which branching events have occurred. To

make such estimates, the timing of one or more divergence events must be confidently estimated. For example, the fossil record may indicate that two clades diverged from a common ancestor at a particular time. Alternatively, the timing of separation of two clades may be estimated from geological events that likely led to their divergence, such as the rise of a mountain that now separates the two clades. With this information, the amount of DNA divergence separating two clades can be divided by the length of time separating the two clades, which produces an estimate of the rate of DNA divergence per unit of time (usually, per million years). Assuming a molecular clock, this rate can then be used to date other divergence events in a cladogram. Although the molecular clock appears to hold true in some cases, in many others the data indicate that rates of evolution have not been constant through time across all branches in an evolutionary tree. For this reason, evolutionary dates derived from molecular data must be treated cautiously. Recently, methods have been developed to date evolutionary events without assuming that molecular evolution has been clocklike. These methods hold great promise for providing more reliable estimates of evolutionary timing.

Learning Outcomes Review 23.2

In cladistics, derived character states are distinguished from ancestral character states, and species are grouped based on shared derived character states. Derived characters are determined from comparison to a group known to be closely related, termed an outgroup. A clade contains all descendants of a common ancestor. A cladogram is a hypothetical representation of evolutionary relationships based on derived character states. Homoplasies may give a false picture of relationships. ■■ ■■

Why is cladistics more successful at inferring phylogenetic relationships in some cases than in others? Why are only shared derived, instead of all derived, characters useful in cladistics for reconstructing phylogenies?

chapter

23 Systematics, Phylogenies, and Comparative Biology 489

23.3 Systematics and Classification Learning Outcomes 1. Explain the taxonomic classification system. 2. Differentiate among monophyletic, paraphyletic, and polyphyletic groups. 3. Explain the meaning of the PSC and why it is controversial.

Whereas systematics is the reconstruction and study of evolutionary relationships, classification refers to how we place species and higher groups—genus, family, class, and so forth—into the taxonomic hierarchy.

The taxonomic classification system is hierarchical Taxonomy is the science of classifying living things. By agreement among taxonomists throughout the world, the first word of the binomial name is the genus to which the organism belongs. This word is always capitalized. The second word refers to the particular species and is not capitalized. The two words together are called the species name (or scientific name) and are written in italics—for example, Homo sapiens. Once a genus has been used in the body of a text, it is often abbreviated in later uses. For example, the dinosaur Tyrannosaurus rex becomes T. rex. Also by agreement among taxonomists, no two plant, animal, or microbial species can have the same scientific name. The scientific name of an organism is the same anywhere in the world and avoids confusion caused by common names that may differ from one place to the next (oddly, the same scientific name can be used for species in different kingdoms, but such duplication is very rare). The taxonomic system is hierarchical, with taxa at a lower level grouped into a smaller number of taxa in the next higher level. Just as species are grouped into genera, genera with similar characteristics are grouped into families, and similar families are placed into the same order. Orders with common properties are placed into the same class, and classes with similar characteristics into the same phylum (plural, phyla). Finally, the phyla are assigned to one of several great groups, the kingdoms. In addition, an eighth level of classification, called a domain, is now frequently used. Figure 23.5 illustrates the classification system, using the gray squirrel as an example. The categories at the different levels may include many, a few, or only one taxon. For example, there is only one living genus of the family Ornithorhynchidae (Ornithorhynchus, containing the species O. anatinus, the duck-billed platypus), but several living genera of Fagaceae (the birch family). To someone familiar with classification or having access to the appropriate reference books, each taxon implies both a set of characteristics and a group of organisms belonging to the taxon.

Current classification sometimes does not reflect evolutionary relationships Systematics and traditional classification are not always congruent; to understand why, we need to consider how species may be 490 part IV Evolution

grouped based on their phylogenetic relationships. A monophyletic group includes the most recent common ­ancestor of the group and all of its descendants. By definition, a clade is a monophyletic group. A paraphyletic group includes the most recent common ancestor of the group, but not all its descendants, and a polyphyletic group does not include the most recent common ancestor of all members of the group (figure 23.6). Taxonomic hierarchies are based on shared traits, and ideally they should reflect evolutionary relationships. Traditional taxonomic groups, however, do not always fit well with the new understanding of phylogenetic relationships. For example, birds have historically been placed in the class Aves, and dinosaurs have been considered part of the class Reptilia. But recent phylogenetic advances make clear that birds evolved from dinosaurs. The last common ancestor of all birds and a dinosaur was a meat-eating dinosaur (figure 23.6). Therefore, having two separate monophyletic groups, one for birds and one for reptiles (including dinosaurs and crocodiles, as well as lizards, snakes, and turtles), is not possible. And yet the terms Aves and Reptilia are so familiar and well established that suddenly referring to birds as a type of dinosaur, and thus a type of reptile, is difficult for some. Nonetheless, biologists increasingly refer to birds as a type of dinosaur and hence a type of reptile. Situations like this are not uncommon. Another example concerns the classification of plants. Traditionally, three major groups were recognized: green algae, bryophytes, and vascular plants (figure 23.7). However, recent research reveals that neither the green algae nor the bryophytes constitutes a mono­phy­let­ic group. Rather, some bryophyte groups are more closely related to vascular plants than they are to other bryophytes, and some green algae are more closely related to bryophytes and vascular plants than they are to other green algae. As a result, systematists no longer recognize green algae or bryophytes as evolutionary groups, and the classification system has been changed to reflect evolutionary relationships.

The phylogenetic species concept focuses on shared derived characters In chapter 22, you read about a number of different ideas concerning what determines whether two populations belong to the same species. The biological species concept (BSC) defines species as groups of interbreeding populations that are reproductively isolated from other groups. In recent years, a phylogenetic perspective has emerged and has been applied to the question of species concepts. Advocates of the phylogenetic species concept (PSC) propose that the term species should be applied to groups of populations that have been evolving independently of other groups of populations. Moreover, they suggest that phylogenetic analysis is the way to identify such species. In this view, a species is a population or set of populations characterized by one or more shared derived character(s)—the rationale being that the possession of a derived character implies a period of separate evolution. This approach solves two of the problems with the BSC that were discussed in chapter 22. First, the BSC cannot be applied to allopatric populations because scientists cannot determine whether individuals of the populations would interbreed and produce fertile offspring if they ever came together. The PSC solves this problem: instead of trying to predict what would happen in the

Domain Eukarya

Kingdom Animalia

Figure 23.5 The hierarchical system used in classifying an organism. The

Phylum Chordata

organism, in this case the eastern gray squirrel, is first recognized as a eukaryote (domain Eukarya). Within this domain, it is an animal (kingdom Animalia). Among the different phyla of animals, it is a vertebrate (phylum Chordata, subphylum Vertebrata). The organism’s fur characterizes it as a mammal (class Mammalia). Within this class, it is distinguished by its gnawing teeth (order Rodentia). Next, because it has four front toes and five back toes, it is a squirrel (family Sciuridae). Within this family, it is a tree squirrel (genus Sciurus), with gray fur and white-tipped hairs on the tail (species Sciurus carolinensis, the eastern gray squirrel).

Subphylum Vertebrata

Class Mammalia

Order Rodentia

future if allopatric populations ever came into contact (that is, would the populations be reproductively isolated?), the PSC looks to the past to determine whether a population (or groups of populations) has evolved independently for a long enough time to develop its own derived characters. Second, the PSC can be applied equally well to both sexual and asexual species, in contrast to the BSC, which deals only with sexual forms.

Family Sciuridae

Genus Sciurus

The phylogenetic species concept also has drawbacks Species Sciurus carolinensis

Sciurus carolinensis

The PSC is controversial, however, for several reasons. First, some critics contend that it will lead to the recognition of every slightly differentiated population as a distinct species. In M ­ issouri, for example, open, desertlike habitat patches called glades are distributed throughout much of the state. These glades contain a chapter

23 Systematics, Phylogenies, and Comparative Biology 491

Archosaurs Giraffe

Bat

Turtle

Crocodile

Stegosaurus

Tyrannosaurus

Velociraptor

Hawk

Monophyletic Group

a.

Dinosaurs

Giraffe

Bat

Turtle

Crocodile

Stegosaurus

Tyrannosaurus

Velociraptor

Hawk

Velociraptor

Hawk

Paraphyletic Group

b. Flying Vertebrates Giraffe

Bat

Turtle

Crocodile

Stegosaurus

Tyrannosaurus

Figure 23.6 Monophyletic, paraphyletic, and polyphyletic groups.  a. A monophyletic group consists

of the most recent common ancestor and all of its descendants. For example, the name “Archosaurs” is given to the monophyletic group that includes crocodiles, Stegosaurus, Tyrannosaurus, Velociraptor, and a hawk. b. A paraphyletic group consists of the most recent common ancestor and some of its descendants. For example, some, but not all, taxonomists traditionally give the name “dinosaurs” to the paraphyletic group that includes Stegosaurus, Tyrannosaurus, and Velociraptor. This group is paraphyletic because one descendant of the most recent ancestor of these species, birds, is not included in the group. Other taxonomists include birds within the Dinosauria because Tyrannosaurus and Velociraptor are more closely related to birds than to other dinosaurs. c. A polyphyletic group does not contain the most recent common ancestor of the group. For example, bats and birds could be classified in the same group, which we might call “flying vertebrates,” because they have similar shapes, anatomical features, and habitats. However, their similarities reflect convergent evolution, not common ancestry.

Polyphyletic Group

c.

?

Inquiry question Based on this phylogeny, are there any alternatives to convergence to explain the presence of wings in birds and bats? What types of data might be used to test these hypotheses?

variety of warmth-loving species of plants and animals that do not occur in the forests that separate the glades. Glades have been isolated from one another for a few thousand years, allowing enough time for populations on each glade to evolve differences in some rapidly evolving parts of the genome. Does that mean that each of the hundreds, if not thousands, of Missouri glades contains its own species of lizards, grasshoppers, and scorpions? Some scientists argue that if one took the PSC to its logical extreme, that is exactly what would result. 492 part IV Evolution

A second problem is that species may not always be monophyletic, contrary to the definition of some versions of the PSC. Consider, for example, a species composed of five populations, with evolutionary relationships like those indicated in figure 23.8. Suppose that population C became isolated and evolved differences that made it qualify as a species by any concept (for example, reproductively isolated, ecologically differentiated). But this distinction would mean that the remaining populations, which might still be perfectly capable of exchanging genes, would be paraphyletic,

Red Algae

Green Algae Chlorophytes

Bryophytes

Charophytes

Liverworts

Hornworts

Vascular Plants Mosses

Figure 23.7 Phylogenetic information transforms plant classification. The traditional classification included two groups that we now realize are not monophyletic: the green algae and bryophytes. For this reason, plant systematists have developed a new classification of plants that does not include these groups (discussed in chapter 29). rather than monophyletic. Such situations probably occur often in the natural world. Phylogenetic species concepts, of which there are many different permutations, are increasingly used, but are also contentious for the reasons just discussed. Evolutionary biologists are trying to find ways to reconcile the historical perspective of the PSC with the process-oriented perspective of the BSC and other species concepts. A

B

C

D

E

Learning Outcomes Review 23.3

The traditional classification is hierarchical, placing each species into a nested set of taxonomic categories. This classification is not always consistent with modern understanding of phylogenetic relationships. By definition, a clade is monophyletic. A paraphyletic group contains the most recent common ancestor, but not all its descendants; a polyphyletic group does not contain the most recent common ancestor of all members. The PSC focuses on the possession of shared derived characters, in contrast to the BSC, which emphasizes reproductive isolation. The PSC solves some problems of the BSC but has difficulties of its own. ■■

Under the BSC, is it possible for a species to be polyphyletic?

23.4 Phylogenetics and

Comparative Biology

Learning Outcomes Figure 23.8 Paraphyly and PSC.  The five populations

initially were all members of the same species, with their historical relationships indicated by the cladogram. Then, population C evolved in some ways to become greatly differentiated ecologically and reproductively from the other populations. By all species concepts, this population would qualify as a different species. However, the remaining four species do not form a clade; they are paraphyletic because population C has been removed and placed in a different species. This scenario may occur commonly in nature, but most versions of the PSC do not recognize paraphyletic species.

1. Explain the importance of homoplasy for interpreting patterns of evolutionary change. 2. Describe how phylogenetic trees can reveal the existence of homoplasy. 3. Discuss how a phylogenetic tree can indicate the timing of species diversification.

 

Phylogenies not only provide information about evolutionary relationships among species, but also are indispensable for understanding how evolution has occurred. By examining the distribution of traits chapter

23 Systematics, Phylogenies, and Comparative Biology 493

Figure 23.9 Parental care in dinosaurs and crocodiles a. Fossil

a.

b.

dinosaur incubating its eggs. This re­markable fossil of Oviraptor shows the dinosaur sitting on its nest of eggs just as chickens do today. Not only is the dinosaur squatting on the nest, but its forelimbs are outstretched, perhaps to shade the eggs. b. A mother exhibiting parental care. Female crocodilians build nests and then remain nearby, guarding them, while the eggs incubate. When they are ready to hatch, the baby crocodiles vocalize; females respond by digging up the eggs and carrying the babies to the water. (a) ©David Clark/

Dinosaurs Alive/Giant Screen Films; (b) Shah, Anup/Nature Picture Library/Alamy Stock Photo

among species in the context of their phylogenetic relationships, much can be learned about how and why evolution has proceeded. In this way, phylogenetics is the basis of all comparative biology.

Homologous features are derived from the same ancestral source; homoplastic features are not In chapter 21, we pointed out that homologous structures are those that are derived from the same body part in a common ancestor. Thus, the forelegs of a dolphin (flipper) and of a horse (leg) are homologous because they are derived from the same bones in an ancestral vertebrate. By contrast, the wings of birds and those of dragonflies are homoplastic structures because they are not derived from the same structure in a common ancestor. Phylogenetic analysis can help determine whether structures are homologous or homoplastic.

Homologous parental care in dinosaurs, crocodiles, and birds Recent fossil discoveries have revealed that many species of dinosaurs exhibited parental care. They incubated eggs laid in nests and took care of the growing baby dinosaurs, many of which could not have fended for themselves. Some recent fossils show dinosaurs sitting on a nest in exactly the same posture used by birds today (figure 23.9a)! Initially, these discoveries were treated as remarkable and unexpected—dinosaurs apparently had independently evolved behaviors similar to those of modern-day organisms. But examination of the phylogenetic position of dinosaurs (see figure 23.6) indicates that they are most closely related to two living groups of animals—crocodiles and birds—both of which exhibit parental care (figure 23.9b). It appears likely, therefore, that the parental care exhibited by crocodiles, dinosaurs, and birds did not evolve convergently from different ancestors that did not exhibit parental care; rather, the behaviors are homologous, inherited by each of these groups from their common ancestor that cared for its young.

?

Inquiry question What if only some types of dinosaurs

exhibited parental care? In light of the phylogeny in figure 23.6, how would that change our inferences about the evolution of parental care?

494 part IV Evolution

Homoplastic convergence: saber teeth and plant conducting tubes In other cases, by contrast, phylogenetic analysis can indicate that similar traits have evolved independently in different clades. This convergent evolution from different ancestral sources indicates that such traits represent homoplasies. As one example, the fossil record reveals that extremely elongated canine teeth (saber teeth) occurred in a number of different groups of extinct carnivorous mammals. Although how these teeth were actually used is still debated, all saber-toothed carnivores had body proportions similar to those of cats, which suggests that these different types of carnivores all evolved into a similar predatory lifestyle. Examination of the sabertoothed character state in a phylogenetic context reveals that it most likely evolved independently at least four times (figure 23.10). Conducting tubes in plants provide a similar example. The tracheophytes, a large group of land plants discussed in chapter 29, transport photosynthetic products, hormones, and other molecules over long distances through elongated, tubular cells that have perforated walls at the end. These structures are stacked upon each other to create a conduit called a sieve tube. Sieve tubes facilitate long-distance transport that is essential for the survival of tall plants on land. Most members of the brown algae, which includes kelp, also have sieve elements (see figure 23.11 for a comparison of the sieve plates in brown algae and angiosperms) that aid in the rapid transport of materials. The land plants and brown algae are distantly related (figure 23.11), and their last common ancestor was a single-celled organism that could not have had a multicellular transport system. This indicates that the strong structural and functional similarity of sieve elements in these plant groups is an example of convergent evolution.

Complex characters evolve through a sequence of evolutionary changes Most complex characters do not evolve, fully formed, in one step. Rather, they are often built up, step-by-step, in a series of evolutionary transitions. Phylogenetic analysis can help discover these evolutionary sequences.

SCIENTIFIC THINKING Question: How many times have saber teeth evolved in mammals? Hypothesis: Saber teeth are homologous and have only evolved once in mammals (or, conversely, saber teeth are convergent and have evolved multiple times in mammals). Phylogenic Analysis: Examine the distribution of saber teeth on a phylogeny of mammals, and use parsimony to infer the history of saber tooth evolution (note that not all branches within marsupials and placentals are shown on the phylogeny).

Saber-toothed nimravid

Saber-toothed nimravid

Hyenas

Saber-toothed cat

Civets

Mongooses

Bears, seals, weasels, canids, and raccoons

Saber-toothed creodont

Phylogeny of Carnivores and Creodonts

Creodonts

Carnivores

Saber-toothed marsupial

Phylogeny of Mammals

Felines Marsupials

Placentals

Nimravids

Monotremes

Carnivores

Creodonts

Result: Saber teeth have evolved at least four times in mammals: once within marsupials, once in felines, once in an extinct group called creodonts, and at least once in another group of now-extinct catlike carnivores called nimravids. Interpretation: Note that it is possible that saber teeth evolved twice in nimravids, but another possibility that requires the same number of evolutionary changes (and thus is equally parsimonious) is that saber teeth evolved only once in the ancestor of nimravids and then were subsequently lost in one group of nimravids. (Note that for clarity, not all branches within marsupials and placentals are shown in this illustration.)

Figure 23.10 Distribution of saber-toothed mammals.

Stramenopiles

Alveolates

Red Algae

Chlorophytes

Chlorophytes

Liverworts

Hornworts

Mosses

Tracheophytes

2 μm

60 μm Brown Alga

Angiosperms

Figure 23.11 Convergent evolution of conducting tubes. Sieve tubes, which transport hormones and other substances throughout the plant, have evolved in two distantly related plant groups (brown algae are stramenopiles and angiosperms are tracheophytes).

(Left) ©Lee W. Wilcox; (Right) Clouds Hill Imaging Ltd./Getty Images

chapter

23 Systematics, Phylogenies, and Comparative Biology 495

Other dinosaurs

Coelophysis

Tyrannosaurus

Sinosauropteryx

Velociraptor

Caudipteryx

Archaeopteryx

Modern Birds

Loss of teeth and reduction of tail Arms longer than legs Long, aerodynamic feathers Long arms, highly mobile wrist, feathers with vanes, shafts, and barbs Downy feathers Wishbone, breastbone, loss of fingers 4 and 5 Light bones

Figure 23.12 The evolution of birds.  The traits we think of as characteristic of modern birds have evolved in stages over many millions of years.

Modern-day birds—with their wings, feathers, light bones, and breastbone—are exquisitely adapted flying machines. Fossil discoveries in recent years now allow us to reconstruct the evolution of these features. When the fossils are arranged phylogenetically, it becomes clear that the features characterizing living birds did not evolve simultaneously. ­­Figure  23.12 shows how the features important to flight evolved sequentially, probably over a long period of time, in the ancestors of modern birds. One important finding often revealed by studies of the evolution of complex characters is that the initial stages of a character evolved as an adaptation to some environmental selective pressure different from that for which the character is currently adapted. Examination of figure 23.12 reveals that the first feathery structures evolved deep in the theropod phylogeny, in animals with forearms clearly not modified for flight. Therefore, the initial feather-like structures must have evolved for some other reason, perhaps serving as insulation or decoration. Through time, these structures have become modified to the extent that modern feathers produce excellent aerodynamic performance.

Phylogenetic methods can be used to distinguish between competing hypotheses Understanding the causes of patterns of biological diversity observed today can be difficult because a single pattern often could have resulted from several different processes. In many cases, scientists can use phylogenies to distinguish between competing hypotheses. 496 part IV Evolution

Larval dispersal in marine snails An example of this use of phylogenetic analysis concerns the evolution of larval forms in marine snails. Most species of snails produce microscopic larvae that drift in the ocean currents, sometimes traveling hundreds or thousands of miles before becoming established and transforming into adults. Some species, however, have evolved larvae that settle to the ocean bottom very quickly and thus don’t disperse far from their place of origin. Studies of fossil snails indicate that the proportion of species that produce nondispersing larvae has increased through geological time (figure 23.13). 40 MYA Eocene

45 MYA 50 MYA 55 MYA

Paleocene

60 MYA 65 MYA 0

50 Percentage of species with nondispersing larvae

100

Figure 23.13 Larval dispersal. Increase through time in the proportion of species whose larvae do not disperse far from their place of birth.

Two processes could produce an increase in nondispersing larvae through time. First, if evolutionary change from dispersing to nondispersing occurred more often than change in the opposite direction, then the proportion of species that are nondispersing would increase through time. Alternatively, if species that were nondispersing speciated more frequently, or became extinct less frequently, than dispersing species, then through time the proportion of nondispersers would also increase (assuming that the descendants of nondispersing species also were nondispersing). This latter case is a reasonable hypothesis because nondispersing species probably have lower amounts of gene flow than dispersing species, and thus might more easily become geographically isolated, increasing the likelihood of allopatric speciation (see chapter 22). These two processes would result in different phylogenetic patterns. If evolution from a dispersing ancestor to a nondispersing descendant occurred more often than the reverse, then an excess of such changes should be evident in the phylogeny, as shown by more transitions from dispersing to nondispersing larvae (indicated by pluses)

than changes in the opposite direction (indicated by minuses) in figure 23.14a. In contrast, if nondispersing species underwent greater speciation, then clades of nondispersing species would contain more species than clades of dispersing species, as shown in figure 23.14b. Evidence for both processes was revealed in an examination of the phylogeny of marine snails in the genus Conus, in which 30% of species are nondispersing (figure 23.14c). The phylogeny indicates that possession of dispersing larvae was the ancestral state; nondispersing larvae are inferred to have evolved eight times, with no evidence for evolutionary reversal from nondispersing to dispersing larvae. At the same time, clades of nondispersing larvae tend to have on average 3.5 times as many species as dispersing larvae, which suggests that in nondispersing species, rates of speciation are higher, rates of extinction are lower, or both. This analysis therefore indicates that the evolutionary increase in nondispersing larvae through time may be a result both of a bias in the direction in which evolution proceeds and of an increase in rate of diversification (that is, speciation rate minus extinction rate) in nondispersing clades.

+ +

–

–

–

+ + +

+ Dispersing larvae Nondispersing larvae

a.

b.

+

+ +

+ + +

+

+

Nondispersing larvae

Dispersing larvae

c.

Figure 23.14 Phylogenetic investigation of the evolution of nondispersing larvae. a. In this hypothetical example, the

evolutionary transition from dispersing to nondispersing larvae occurs more frequently (four times, indicated by a plus sign) than the converse (once, indicated by a minus sign). By contrast, in (b) the number of evolutionary changes in each direction is the same, but clades that have nondispersing larvae diversify to a greater extent due to higher rates of speciation or lower rates of extinction (assuming that extinct forms are not shown). c. Phylogeny for Conus, a genus of marine snails. Nondispersing larvae have evolved eight separate times from dispersing larvae, with no instances of evolution in the reverse direction. This phylogeny does not show all species, however; nondispersing clades contain on average 3.5 times as many species as dispersing clades. chapter

23 Systematics, Phylogenies, and Comparative Biology 497

+

+

+

+ +

+ +

+

+

+

+ +

+

+

+

+

+

+

a.

?

+ +

+ +

Direct development Larval stage

–

–

–

+

Figure 23.15 Evolution of direct development in a family of limpets. a. Direct

b.

development evolved many times (beige lines stemming from a red ancestor, indicated by a plus sign), and three instances of reversed evolution from direct development to larval development are indicated (red lines from a beige ancestor, indicated by a minus sign). b. A less parsimonious interpretation of evolution in the clade in the light blue box is that, rather than two evolutionary reversals, six instances of the evolution of development occurred without any evolutionary reversal.

Inquiry question How might you distinguish between the hypotheses in parts a and b of this figure?

The lack of evolutionary reversal is not surprising because when larvae evolve to become nondispersing, they often lose a variety of structures used for feeding while drifting in the ocean current. In most cases, once a structure is lost, it rarely re-evolves, and thus the standard view is that the evolution of nondispersing larvae is a oneway street, with few examples of re-evolution of dispersing larvae.

Loss of the larval stage in marine invertebrates A related phenomenon in many marine invertebrates is the loss of the larval stage entirely. Most marine invertebrates—in groups as diverse as snails, sea stars, and anemones—pass through a larval stage as they develop. But in a number of different types of organisms, the larval stage is omitted, and the eggs develop directly into adults. The evolutionary loss of the larval stage has been suggested as another example of a nonreversible evolutionary change because once the larval stages are lost, it is difficult for them to re-evolve— or so the argument goes. A recent study on one group of marine limpets, shelled marine organisms related to snails, shows that this is not necessarily the case. Among these limpets, direct development has evolved many times; however, in three cases, the phylogeny strongly suggests that evolution reversed and a larval stage re-evolved (figure 23.15a). It is important to remember that patterns of evolution suggested by phylogenetic analysis are not always correct—evolution does not necessarily occur parsimoniously. In the limpet study, for example, it is possible that within the clade in the light blue box, presence of a larva was retained as the ancestral state, and direct development evolved independently six times (figure 23.15b). Phylogenetic analysis cannot rule out this possibility, even if it is less phylogenetically parsimonious. If the re-evolution of lost traits seems unlikely, then the alternative hypothesis that direct development evolved six times—rather than only once at the base of the clade, with two instances of evolutionary 498 part IV Evolution

reversal—should be considered. For example, studies of the morphology or embryology of direct-developing species might shed light on whether such structures are homologous or convergent. In some cases, artificial selection experiments in the laboratory or genetic manipulations can test the hypothesis that it is difficult for lost structures to reevolve. Conclusions from phylogenetic analyses are always stronger when supported by results of other types of studies.

Phylogenetics helps explain species diversification One of the central goals of evolutionary biology is to explain patterns of species diversity: Why do some types of plants and animals exhibit more species richness—a greater number of species per clade—than others? Phylogenetic analysis can be used both to suggest and to test hypotheses about such differences.

Species richness in beetles Beetles (order Coleoptera) are the most diverse group of animals. Approximately 60% of all animal species are insects, and there are far more species of beetles than of any other type of insect. Among beetles, families that are herbivorous are particularly species-rich. Examination of the phylogeny provides insight into beetle evolutionary diversification (figure 23.16). Among the Phytophaga, the clade which contains most herbivorous beetle species, the deepest branches belong to beetle families that specialize on conifers. This finding agrees with the fossil record because conifers were among the earliest seed plant groups to evolve. By contrast, the flowering plants (angiosperms) evolved more recently, in the Cretaceous, and beetle families specializing on them have shorter evolutionary branches, indicating their more recent evolutionary appearance. This correspondence between phylogenetic position and timing of plant origins suggests that beetles have been remarkably

33,400

18

8

400

3

25,000

78

150

20

10

41,602

1500

24

2000

85

Number of Species Tertiary

23.5

Phylogenetics and Disease Evolution

Learning Outcome

Cretaceous

1. Discuss how phylogenetic analysis can help identify patterns of disease transmission. Jurassic

Triassic

Conifer Cycad Angiosperm

Figure 23.16 Evolutionary diversification of the Phytophaga, the largest clade of herbivorous beetles. 

Clades that originated deep in the phylogenetic tree feed on conifers; clades that feed on angiosperms, which evolved more recently, originated more recently. Age of clades is established by examination of fossil beetles.

The examples so far have illustrated the use of phylogenetic analysis to examine relationships among species. Such analyses can also be conducted on virtually any group of biological entities, as long as evolutionary divergence in these groups occurs by a branching process, with little or no genetic exchange between different groups. No example illustrates this better than recent attempts to understand the evolution of viruses. In this section, we will first see how phylogenetic analysis has been important in discovering the origin and evolution of HIV, the virus that causes acquired immunodeficiency syndrome (AIDS). Then, we will see how the same approach has proven critical in understanding the recent coronavirus pandemic.

conservative in their diet. The family Nemonychidae, for example, appears to have remained specialized on conifers since the beginning of the Jurassic, approximately 210 mya.

HIV has evolved from a viral counterpart present in another primate species

Phylogenetic explanations for beetle diversification

AIDS was first recognized in the early 1980s, and it rapidly became epidemic in the human population. Current estimates are that more than 38 million people are infected with the human immunodeficiency virus (HIV), of whom nearly 700 thousand die each year. At first, scientists were perplexed about where HIV had originated and how it had infected humans. In the mid-1980s, however, scientists discovered a related virus in laboratory ­monkeys, termed simian immunodeficiency virus (SIV). In b­iochemical terms, the viruses are very similar, although genetic differences exist. At last count, SIV has been detected in more than 40 species of primates, but only in species found in sub-Saharan Africa. Interestingly, SIV—which appears to be transmitted sexually—does not appear to cause illness in some of these species. Based on the degree of genetic differentiation among strains of SIV, scientists estimate that SIV may have been around for more than a million years in these primates, perhaps providing enough time for these species to adapt to the virus and thus prevent it from having adverse effects.

The phylogenetic perspective suggests factors that may be responsible for the incredible diversity of beetles. The phylogeny for the Phytophaga indicates that it is not the evolution of herbivory itself that is linked to great species richness. Rather, specialization on angiosperms seems to have been a prerequisite for great species diversification. Specialization on angiosperms appears to have arisen five times independently within herbivorous beetles; in each case, the angiosperm-specializing clade is substantially more speciesrich than the clade to which it is most closely related (termed a sister clade) and which specializes on some other type of plant. Why specialization on angiosperms has led to great species diversity is not yet clear and is the focus of much current research. One possibility is that this diversity is linked to the great speciesrichness of angiosperms themselves. With more than 300,000 species of angiosperms, beetle clades specializing on them may have had a multitude of opportunities to adapt to feed on different species, thus promoting divergence and speciation.

Learning Outcomes Review 23.4

Homologous traits are derived from the same ancestral character states, whereas homoplastic traits are not, even though they may have similar function. Phylogenetic analysis can help determine whether homology or homoplasy has occurred. By correlating phylogenetic branching with known evolutionary events, the timing and cause of diversification can be inferred. ■■

 

Does the possession of the same character state by all members of a clade mean that the ancestor of that clade necessarily possessed that character state?

Phylogenetic analysis identifies the path of transmission Phylogenetic analysis of strains of HIV and SIV reveals three clear findings. First, HIV obviously descended from SIV. All strains of HIV are phylogenetically nested within clades of SIV strains, indicating that HIV is derived from SIV (figure 23.17). Second, a number of different strains of HIV exist, and they appear to represent independent transfers from different primate species. Each of the human strains is more closely related to a strain of SIV than it is to other HIV strains, indicating separate origins of the HIV strains. chapter

23 Systematics, Phylogenies, and Comparative Biology 499

SIV- De Brazza’s monkey

SIV- greater spot-nasal monkey

SIV- Syke’s monkey

SIV- sooty mangabey

HIV- 2 group B

HIV- 2 group A

SIV- sooty mangabey

SIV- tantalus

SIV- vervet monkey

SIV- drill

SIV- red-capped mangabey

SIV- chimpanzee

SIV- gorilla

HIV- 1 group P

HIV- 1 group O

SIV- chimpanzee

SIV- chimpanzee

SIV- chimpanzee

SIV- chimpanzee

HIV- 1 group N

SIV- chimpanzee

HIV- 1 group M

relatives, are plummeting toward extinction. A second consequence of this hunting is that humans are increasingly brought into contact with bodily fluids of these animals, and it is easy to imagine how during the butchering process, blood from a recently killed animal might enter the human bloodstream through open sores or cuts in the skin.

Establishing the crossover time line and location

Where and when did this cross-species transmission occur? HIV strains are most diverse in Africa, and the incidence of HIV is higher there than elsewhere in the world. Combined with the evidence that HIV is related to SIV in African primates, it seems certain that AIDS appeared first in Africa. As for when the jump from other primates to humans occurred, the fact that AIDS was not recognized until the 1980s suggests that HIV probably arose recently. Descendants of slaves brought to North America from West Africa in the 19th century lacked Indicates the disease, indicating that it probably did transmission to humans not occur at the time of the slave trade. Once the disease was recognized in the 1980s, scientists scoured repositories of old Figure 23.17 Evolution of HIV and SIV. HIV has evolved multiple times and from blood samples to see whether HIV could be strains of SIV in different primate species (each primate species indicated by a different detected in blood samples from the past. The color; drill and tantalus are species of monkey). Red rectangles indicate transmission to earliest HIV-positive result was found in a humans from other primate species. sample from 1959, pushing the date of origin back at least two decades. Based on the amount of genetic difference between strains of HIV-1, including Finally, humans have acquired HIV from different host spethe 1959 sample, and assuming the operation of a molecular clock, cies. HIV-1, which is the virus responsible for the global epidemic, scientists estimate that the deadly strain of AIDS probably crossed has four subtypes. Two of these subtypes are most closely related into humans in the 1920s. to strains in chimpanzees, whereas a third is most closely related to a strain in gorillas, indicating transmission from both ape species. The origin of the fourth subtype is not clear; further samPhylogenies can be used to track the evolution pling will likely reveal it to be the sister taxon to a currently of AIDS among individuals undiscovered chimp or gorilla strain. The AIDS virus evolves extremely rapidly, so much so that difBy contrast, subtypes of HIV-2, which is much less wideferent strains can exist within a single individual in a single popuspread (in some cases known from only one individual), are related lation. As a result, phylogenetic analysis can be applied to to SIV found in West African monkeys, primarily the sooty manganswer very specific questions; just as phylogeny proved useful abey (Cercocebus atys). Moreover, the subtypes of HIV-2 also apin determining the source of HIV, it can also pinpoint the source pear to represent multiple cross-species transmissions to humans. of infection for particular individuals. Transmission from other primates to humans This ability became apparent in a court case in Louisiana in Several hypotheses have been proposed to explain how SIV jumped 1998, in which a dentist was accused of injecting his former girlfrom chimps and monkeys to humans. The most likely idea is that friend with blood drawn from an HIV-infected patient. The dentist’s transmission occurred as the result of blood-to-blood contact that records revealed that he had drawn blood from the patient and had may occur when humans kill and butcher monkeys and apes. Recent done so in a suspicious manner. Scientists sequenced the viral strains years have seen a huge increase in the rate at which primates are from the victim, from the patient, and from a large number of HIVhunted for the “bushmeat” market, particularly­in central and westinfected people in the local community. The phylogenetic analysis ern Africa. This increase has resulted from growing human populaclearly demonstrated that the victim’s viral strain was most closely tions desiring greater amounts of protein combined with increased related to the patient’s (figure 23.18). This analysis, which for the access to the habitats in which these primates live as the result of first time established phylogenetics as a legally admissible form of road building and economic development. The unfortunate result is evidence in courts in the United States, helped convict the dentist, that population sizes of many primate species, including our closest who is now serving a 50-year sentence for attempted murder. 500 part IV Evolution

Community

Patient

Victim

Figure 23.18 Evolution of HIV strains reveals the source of infection.  HIV mutates so rapidly that multiple genotypes often circulate within the body of a single person infected with HIV. As a result, it is possible to create a phylogeny of HIV strains and to identify the source of infection of a particular individual. In this case, the HIV strains of the victim (pink) clearly are derived from strains in the body of another individual, the patient (blue). Other HIV strains are from HIV-infected individuals in the local community.

?

Inquiry question What would the phylogeny look like if the victim had not contracted HIV from the patient?

California early in the pandemic indicated that already by March 2020, at least seven different strains had arrived in the state, indicating introduction of the disease from multiple localities on several continents. On the other hand, clusters of patients possessing the same strain indicated the occurrence of community transmission from one individual to another. A similar phylogenetic analysis of patients in New York City in March 2020 identified at least nine introductions, most from Europe or elsewhere in North America, and multiple cases of community transmission.

Phylogenies also have been used to study the origin and spread of COVID-19 When SARS-CoV-2 (COVID-19) emerged in early 2020, scientists were well prepared to investigate its origin and spread following the approaches established to study HIV and other emerging diseases over the previous several decades. Moreover, the capacity to collect DNA sequence data rapidly and in large quantities had greatly expanded since the emergence of earlier diseases. Very quickly, the virus responsible for the disease was identified as a coronavirus, similar to the one that had caused the SARS outbreak of 2003 (see chapter 26). Phylogenetic analysis indicated that the most closely related viral lineages all occurred in bat species, suggesting that the disease was the result of a virus that had jumped from bats to humans (figure 23.19). One complication was that the disease apparently first emerged in a market in Wuhan, China, where live animals of many species were kept for sale in very crowded conditions. Presumably, the virus had been passed to humans at that market. However, bats were not on sale at the market, so it was suspected that an intermediate species, which had gotten the virus from a bat and then passed it on to humans, was involved. Exactly that scenario happened in 2003 with SARS—genetic analysis identified the civet, a cat-sized Asian carnivorous mammal, as the intermediate species and horseshoe bats as the natural reservoir from which the disease had emerged. The unexpected discovery that closely related viruses occur in the pangolin, an obscure ant-eating species endangered by illegal wildlife trafficking, suggested that this species might be the conduit between bats and humans, but subsequent research has been equivocal. At this point, the intermediate species still has not been conclusively identified and researchers continue the search. In addition, the possibility that the virus escaped from a research laboratory is still under investigation, though evidence supporting this hypothesis is scant. Phylogenetic analysis of genetically different strains of COVID-19 in different patients has been used to understand how the disease spread, just as with HIV. One study among patients in

Learning Outcomes Review 23.5

Modern phylogenetic techniques and analysis can track the evolution of disease strains, uncovering sources and progression. The HIV virus provides a prime example: analysis of viral strains has shown that the progression from simian immunodeficiency virus (SIV) into human hosts has occurred several times. Phylogenetic analysis is also used to track the transmission of human disease. ■■

Could HIV have arisen in humans and then been transmitted to other primate species?

Human

Bat

Bat

Pangolin Pangolin

Bat

Bat

Figure 23.19 Phylogenetic relationships of coronaviruses. The most closely related virus to the one that

causes COVID-19 in humans occurs in bat. Because pangolins harbor a similar, though phylogenetically more distant, virus, some have suggested that pangolins may be the intermediate host that transmitted COVID-19 from bats to humans.  EcoPic/Getty Images chapter

23 Systematics, Phylogenies, and Comparative Biology 501

Chapter Review Imagemore Co, Ltd./Imagemore/Getty Images

23.1 Systematics

Branching diagrams depict evolutionary relationships. Systematics is the study of evolutionary relationships, which are depicted on branching evolutionary trees, called phylogenies. Similarity may not accurately predict evolutionary relationships. The rate of evolution can vary among species and can even reverse direction. Closely related species can therefore be dissimilar in phenotypic characteristics. Conversely, convergent evolution results in the phenotypic similarity of distantly related species.

23.2 Cladistics The cladistic method requires that character variation be identified as ancestral or derived. Derived character states are those that differ from the ancestral condition. Only shared derived characters are useful for inferring phylogenies. Character polarity is established using an outgroup comparison in which the outgroup consists of one or a group of species known to be outside of the group under study. Character states exhibited by the outgroup are assumed to be ancestral, and other character states are considered derived. Homoplasy complicates cladistic analysis. Homoplasy refers to a shared derived character state, such as wings of birds and wings of insects, that has not been inherited from a common ancestor. Cladograms are constructed based on the principle of parsimony, which indicates that the phylogeny requiring the fewest evolutionary changes is accepted as the best working hypothesis. Other phylogenetic methods work better than cladistics in some situations. When evolutionary change is rapid, other methods, such as statistical approaches and the use of the molecular clock, are sometimes more useful.

23.3 Systematics and Classification The taxonomic classification system is hierarchical. Species are grouped into genera, genera into families, and so on through the ranks of order, class, phylum, kingdom, and domain. Current classification sometimes does not reflect evolutionary relationships. A monophyletic group consists of the most recent common ancestor and all of its descendants. A paraphyletic group consists of the most recent common ancestor and some of its descendants. A polyphyletic group does not contain the most recent ancestor of the group. Some currently recognized taxa are not monophyletic, such as reptiles, which are paraphyletic with respect to birds.

502 part IV Evolution

The PSC focuses on shared derived characters. The PSC emphasizes the possession of shared derived characters, whereas the BSC focuses on reproductive isolation. Many versions of the PSC recognize species that are monophyletic. The PSC also has drawbacks. Among criticisms of the PSC are that it recognizes as a distinct species any population with any degree of genetic difference from other populations and that evolution routinely produces paraphyletic groups that would be considered to be species by most other species concepts.

23.4 Phylogenetics and Comparative Biology Homologous features are derived from the same ancestral source; homoplastic features are not. Phylogenetic analysis can indicate whether different structures have been built from the same ancestral structure and thus may be considered homologous. Complex characters evolve through a sequence of evolutionary changes. Most complex features do not evolve in a single step but include stages of transition. They may have begun as an adaptation to a selective pressure different from the one for which the feature is currently adapted. Phylogenetic methods can be used to distinguish between competing hypotheses. Different evolutionary scenarios can be distinguished by phylogenetic analysis. The minimum number of times a trait may have evolved can be established, and the direction of trait evolution, the timing, and the cause of diversification can be inferred. Phylogenetics helps explain species diversification. Questions regarding the causes of species richness may be addressed with phylogenetic analysis.

23.5 Phylogenetics and Disease Evolution HIV has evolved from a viral counterpart present in another primate species. Phylogenetic methods have indicated that HIV is related to SIV. Phylogenetic analysis identifies the path of transmission. It is clear that HIV has descended from SIV, and that independent transfers from other primate species to humans have occurred several times. Phylogenies can be used to track the evolution of AIDS among individuals. Because HIV evolves rapidly, phylogenetic analysis can trace the origin of a current strain to a specific source of infection. Phylogenies also have been used to study the origin and spread of COVID-19. COVID-19 is derived from viruses found in bats. Phylogenetic analysis indicates multiple introductions and community transmission.

Visual Summary Evolutionary relationships Ancestral characters identifies Derived characters

Cladistics

arranged as a

Phylogenies

Classification

used to infer

Homoplasy

Evolution

explains

as a result of

Homology

Hierarchy

depicted by

complicated by

Species with similar features

Homoplasy

inferred by

may not represent

understood by studying

is key to

Comparative biology

Studying disease dynamics

enlightens patterns of

identifies

Species diversification

Path of transmission

tracks

Evolution of HIV and COVID-19

Review Questions Imagemore Co, Ltd./Imagemore/Getty Images

U N D E R S TA N D 1. Overall similarity of phenotypes may not always reflect evolutionary relationships a. b. c. d.

due to convergent evolution. because of variation in rates of evolutionary change of different kinds of characters. due to homoplasy. All of the choices are correct.

2. Cladistics a. b. c. d.

is based on overall similarity of phenotypes. requires distinguishing similarity due to inheritance from a common ancestor from similarity for other reasons. is not affected by homoplasy. None of the choices is correct.

3. The principle of parsimony a. b. c. d.

helps evolutionary biologists distinguish among competing phylogenetic hypotheses. does not require that the polarity of traits be determined. is a way to avoid having to use outgroups in a phylogenetic analysis. cannot be applied to molecular traits.

4. Parsimony suggests that parental care in birds, crocodiles, and some dinosaurs a. b. c. d.

evolved independently multiple times by convergent evolution. evolved once in an ancestor common to all three groups. is a homoplastic trait. is not a homologous trait.

5. The forelimb of a bird and the forelimb of a rhinoceros a. b. c. d.

are homologous and symplesiomorphic. are not homologous but are symplesiomorphic. are homologous and synapomorphic. are not homologous but are synapomorphic.

6. In order to determine polarity for different states of a character a. b. c. d.

there must be a fossil record of the groups in question. genetic sequence data must be available. an appropriate name for the taxonomic group must be selected. an outgroup must be identified.

7. In a paraphyletic group a. b.

all species are more closely related to each other than they are to a species outside the group. evolutionary reversal is common. chapter

23 Systematics, Phylogenies, and Comparative Biology 503

c. d.

polyphyly also usually occurs. some species are more closely related to species outside the group than to some species within the group.

8. A paraphyletic group includes a. b. c. d.

an ancestor and all of its descendants. an ancestor and some of its descendants. descendants of more than one common ancestor. All of the choices are correct.

9. Sieve tubes and sieve elements are a. b. c. d.

homoplastic because they have different function. homologous because they have similar function. homoplastic because their common ancestor was single-celled. structures involved in transport within animals.

A P P LY 1. A taxonomic group that contains species that have similar phenotypes due to convergent evolution is a. paraphyletic. b. monophyletic.

c. polyphyletic. d. a good cladistic group.

2. Rapid rates of character change relative to the rate of speciation pose a problem for cladistics because a. b. c. d.

the frequency with which distantly related species evolve the same derived character state may be high. evolutionary reversals may occur frequently. homoplasy will be common. All of the choices are correct.

3. Species recognized by the PSC a. b. c. d.

sometimes also would be recognized as species by the BSC. are sometimes paraphyletic. are characterized by symplesiomorphies. are more frequent in plants than in animals.

504 part IV Evolution

SYNTHESIZE 1. List the synapomorphy and the taxa defined by that synapomorphy for the groups pictured in figure 23.2. Name each group defined by a set of synapomorphies in a way that might be construed as informative about what kind of characters define the group. 2. Identifying “outgroups” is a central component of cladistic analysis. As described on page 487, a group is chosen that is closely related to, but not a part of, the group under study. If one does not know the relationships of members of the group under study, how can one be certain that an appropriate outgroup is chosen? Can you think of any approaches that would minimize the effect of a poor choice of outgroup? 3. As noted in your reading, cladistics is a widely utilized method of systematics, and our classification system (taxonomy) is increasingly becoming reflective of our knowledge of evolutionary relationships. Using birds as an example, discuss the advantages and disadvantages of recognizing them as reptiles versus as a group separate from and equal to reptiles. 4. Across many species of limpets, loss of larval development and reversal from direct development appear to have occurred multiple times. Under the simple principle of parsimony, are changes in either direction merely counted equally in evaluating the most parsimonious hypothesis? If it is much more likely to lose a larval mode than to re-evolve it from direct development, should that be taken into account? If so, how? 5. Birds, pterosaurs (a type of flying reptile that lived in the Cretaceous period), and bats all have modified their forelimbs to serve as wings, but they have done so in different ways. Are these structures homologous? If so, how can they be both homologous and convergent? Do any organisms possess wings that are convergent, but not homologous? 6. In what sense does the BSC focus on evolutionary mechanisms and the PSC on evolutionary patterns? Which, if either, is correct?

24 CHAPTER

Genome Evolution Chapter Contents 24.1

Comparative Genomics

24.2 Genome Size 24.3 Evolution Within Genomes 24.4 Gene Function and Expression Patterns 24.5 Applying Comparative Genomics

Stephen Robinson/NHPA/Photoshot/Newscom

Introduction

Visual Outline Genomic analysis includes

Comparative genomics

Genome evolution

Genome size

informs reveals Phylogeny

increases due to

Duplication whole genome

Rate of evolution

Functional genomics

acquiring new function

examining

Rearranged DNA

Species-specific genes

and complicated affected by by Noncoding DNA

applications in

and Species-specific expression

Polyploidy Nicotiana tomentosiformis Diploid (TT)

Transposable elements and

ST hybrid Genome duplication Nicotiana tabacum Allopolyploid (SSTT)

Horizontal gene transfer Bacteria

Archaea

Noncoding DNA

Medicine

Eukarya

Conservation biology asts opl l or a Ch ndri ho c ito M

Nicotiana sylvestris Diploid (SS)

Genomes contain the raw material for evolution, and many clues to evolution are hidden in the ever-changing nature of genomes. As more genomes have been sequenced, the new and exciting field of comparative genomics has emerged and has yielded some surprising results as well as many, many questions. Comparing whole genomes, not just individual genes, enhances our ability to understand the workings of evolution, to improve crops, and to identify the genetic basis of disease so that we might develop more effective treatments with minimal side effects. The focus of this chapter is the role of comparative genomics in enhancing our understanding of genome evolution and how this new knowledge can be applied to improve our lives.

24.1 Comparative

Genomics

Learning Outcomes 1. Describe the kinds of differences that can be found between genomes. 2. Explain how we can find human-specific differences. 3. Explain why genomes may evolve at different rates.

A key challenge of modern evolutionary biology is finding a way to link changes in DNA sequences, which we are now able to study in great detail, with the evolution of the complex morphological characters used to construct a traditional phylogeny. Many different genes contribute to complex characters. Making the connection between a specific change in a gene and a modification in a morphological character is particularly difficult. Comparing genomes provides a powerful tool for exploring evolutionary divergence among organisms to connect DNA-level changes with morphological differences. Genomes are more than instruction books for building and maintaining an organism; they also record the history of life. The growing number of fully sequenced genomes in all kingdoms is leading to a revolution in comparative evolutionary biology (figure 24.1). Genetic differences between species can be explored in a very direct way, examining the footprints on the evolutionary path between different species.

Evolution of the vertebrate genome Since the first human genome was sequenced, literally thousands of human genomes have been sequenced. But while this allows inferences about variation within humans, it tells us nothing about the evolution of the human genome. To address this, there has been a large-scale effort to sequence as many chordate genomes as possible. As vertebrates make up the majority of chordates (see chapter 34), this is a very broad data set for comparative genomics. There are literally sequences for hundreds of vertebrates, from aardvark to zebrafish, available. This has allowed the curation of almost 16,000 orthologous genes, that is, genes that are considered to have been inherited from a common ancestor, in vertebrates. This effort is ongoing, and these data are publicly available, which will accelerate vertebrate genomics studies.

Comparison between human and pufferfish genomes It may seem strange that the first animal genome compared to human was the pufferfish (Fugu rubripes), but it was the second vertebrate genome sequenced. These two animals last shared a common ancestor 450 mya. Some human and pufferfish genes have been conserved during evolution, but others are unique to each species. About 25% of human genes have no counterparts in Fugu. Also, extensive genome rearrangements have occurred during the 450 million years since the mammal lineage and the teleost fish diverged, indicating a considerable scrambling of gene order. Finally, the human genome contains about 50% repetitive DNA, but repetitive DNA accounts for less than one-sixth of the Fugu sequence. 506 part IV Evolution

Comparison between human and mouse genomes The vertebrate other than human with the most genomic data is the mouse. The importance of the mouse as a model system has led to equivalent projects for virtually every human genome–related project. A comparison of the genomes reveals that both have about 20,000 protein-coding genes, essentially all of which are shared by both species. There is more divergence in genes that encode long noncoding RNAs (lncRNA), and microRNAs (miRNA), where only 10–20% of these genes appear to be conserved between the two species. As these are either known (miRNA) or thought to be (lncRNA) involved in regulating gene expression, these may explain more of the differences between species than the protein-coding genes.

The protovertebrate genome Studies like those detailed above, and other mapping studies, are being used to reassemble a karyotype for the original protovertebrate genome. These studies take advantage of the fact that we now have genome sequences for the coelocanth, a representative of lobe-finned fish, which gave rise to all vertebrate tetrapods (see chapter 34), and to nonvertebrate chordates, which branched off from the vertebrate lineage prior to any vertebrate-specific rearrangements. One model proposes an ancestral chordate genome that consisted of 17 chromosomes. This ancient genome then underwent a whole-genome duplication event to produce 34  chromosomes. These 34 chromosome then underwent a series of fusion events, followed by a second round of whole-genome duplication, to produce 54 chromosomes in the ancestor to vertebrates. In the lineage leading to amniotes (birds, reptiles, and mammals; see chapter 34), more fusion events occurred leading to 49 chromosomes in the amniote genome. This protoamniote genome has undergone further rearrangements in different lineages to produce the karyotypes we see today.

The human genome shares similarities with those of great apes Genome projects have been completed or are under way for the majority of extant primates. Among these, the chimpanzee, Pan troglodytes, and bonobo, Pan paniscus, share the most recent common ancestor with humans. Comparisons among these genomes reveal 1.4% divergence in single-site substitutions between humans and chimps, and 1.5% between humans and bonobos. There is another 1.5% of the genome that differs in small insertions and deletions (indels) between humans and chimps, and a similar percentage between humans and bonobos. Comparing all sequenced primates indicates that the prevalence of transposable elements observed in humans (chapter 18) is also true for other primates. About 50% of all primate genomes consist of various transposons. Species-specific insertions of these elements varies considerably in different lineages and different elements have expanded in some lineages more than others. In humans, Alu insertions have expanded almost twice as fast as in the chimp and bonobo genomes. As these elements can also facilitate deletions or duplications, they can have effects on genome evolution outside of insertion events. The majority of protein-coding genes found in humans have homologues among both great apes and old-world apes.

Pan troglodytes (chimpanzee)

Fugu rubripes (pufferfish)

Anopheles gambiae (mosquito)

Ornithorynchus anatinus (duck-billed platypus) 2,996 Mb 18,759 genes

393 Mb 18,523 genes Drosophila melanogaster (fruit fly)

265 Mb 14,086 genes

1,918 Mb 21,698 genes

Bos taurus (domestic cow)

Mus musculus (mouse)

Caenorhabditis elegans (a translucent worm) 142 Mb 13,918 genes

3,384 Mb 20,300 genes

2,650 Mb 19,994 genes Schizosaccharomyces pombe (fission yeast)

3,482 Mb 22,606 genes

103 Mb 20,447 genes

Homo sapiens (human)

Zea mays (maize) Oryza sativa (rice)

Arabidopsis thaliana (wall cress)

12.6 Mb 6,991 genes

2 μm Saccharomyces cerevisiae (baker’s yeast)

2,067 Mb 39,475 genes 373 Mb 39,045 genes

120 Mb 35,378 genes

12.2 Mb 5,906 genes

Thermoplasma volcanium (archaea)

25 μm

Plasmodium falciparum (malaria parasite) Escherichia coli (bacteria)

1.6 Mb 1,550 genes

1 μm

23.3 Mb 5,500 genes 4.6 Mb 4,377 genes

8,800×

1 μm

Figure 24.1 Milestones of comparative eukaryotic genomics. The sizes

of genomes listed are the latest found in a variety of genome databases. The number of genes refers specifically to protein coding genes, or so-called coding sequences (CDS). This does not include genes for microRNAs and other forms of RNA.

(mosquito) Source: James Gathany/CDC; (pufferfish) Stephen Frink/Digital Vision/Getty Images; (duck-billed platypus) Paulo Oliveira/Alamy Stock Photo; (chimpanzee) Eric Gevaert/Shutterstock; (a translucent worm) Heiti Paves/Alamy stock photo; (fruit fly) Thomas Deerinck, NCMIR/Getty Images; (mouse) G.K. & Vikki Hart/Photodisc/Getty Images; (domestic cow) Alan and Sandy Carey/Getty Images; (human) James Stevenson/Science Source; (wall cress) Nigel Cattlin/Alamy Stock Photo; (maize) stevanovicigor/iStock/ Getty Images; (rice) Photo by Gary Kramer, U.S. Department of Agriculture Natural Resources Conservation Service; (fission yeast) Steve Gschmeissner/Science Source; (baker’s yeast) Dr. Jeremy Burgess/Science Source; (bacteria) BSIP/age fotostock; (archaea) Dr. Harald Huber and Prof. Dr. Reinhard Rachel, University of Regensburg, Regensburg, Germany; (malaria parasite) T. Brain/Science Source

Type of sites Neanderthal sites Modern human sites

Dates of occupation 30,000–45,000 45,000–135,000 135,000–250,000

Figure 24.2 Europeans and Asians, but not Africans, share between 1% and 4% of their genomes with Neanderthals.  From 30,000 to 45,000 years ago humans and Neanderthals coexisted in Europe, and possibly in the Middle East as early as 80,000 years ago. Interbreeding likely occurred after European and Asian ancestors left Africa.

Despite this, some gene families have experienced lineage-specific expansion and contraction in different lineages. Genes in the major histocompatibility complex (MHC) are expanded in macaques relative to humans, perhaps related to differential exposure to pathogens. Humans and chimps show interesting lineage-specific gains and losses of the zinc finger transcription factor family of genes. The effect of these differences in transcription factors on patterns of gene expression in chimps and humans is not clear, but might potentially be involved in species differences. A recent analysis of lineage-specific differences in structural variation among primates has shown that there are close to 18,000 human-specific structural variants. These include around 12,000 different fixed human-specific insertions, and almost 6,000 fixed human-specific deletions. There is some correlation between the location of these variants and regions known to be involved in regulating gene expression. One tantalizing example is the deletion of a region thought to regulate two cell-cycle genes active in cortical neurons. As one human-specific morphological difference is an enlarged brain, this kind of alteration could lead to more cell division during brain development. All of these functional studies are in their infancy, but they indicate the direction this research is going.

The human genome contains sequences from extinct primates It is now possible to sequence DNA isolated from fossil starting material. This has led to what sounds like a science fiction movie: the Neanderthal reference genome. Yes, we now have sequenced not one, but several genomes from Homo neanderthalensis. The biggest surprise from this work is that our genomes contain the genetic remains of ancient hybridization between humans and Neanderthals. Present-day humans from non-African populations inherited from 1 to 3% of their DNA from Neanderthals 508 part IV Evolution

(figure 24.2). As Neanderthals preceded humans out of Africa into Europe, this must have occurred when early humans and Neanderthals coexisted on the European continent prior to the extinction of Neanderthals around 28,000 years ago. As if this was not surprising enough, DNA isolated from a single finger bone from a cave in the Altai mountains in Siberia, proved to be from a previously unknown hominin we now call Denisovans, for the name of the cave. Populations from Oceania (Australia, Papua New Guinea, and nearby islands) inherited up to 5% of their genomes from Denisovans. Populations on mainland Asia have a much lower percentage of Denisovan DNA. The final surprise was the recent sequencing of another fossil genome that appears to represent an individual with a Denisovan father and a Neanderthal mother. This would be the first such hybrid identified, but it fits with the idea that there was much more interbreeding among these extinct primates than previously thought. In searching for genetic features unique to humans, researchers have taken advantage of the thousands of human genomes sequenced, and the improving quality of the Neanderthal reference genome, to search for human genetic variation missing in Neanderthals. Looking at variation shared by 90% of humans analyzed, 105,757 SNPs and 3,900 indels are unique to humans. For protein-coding genes, there are 96 fixed substitutions in a total of 87 proteins. There are also about 3000 changes that potentially affect gene expression. These results are just a beginning, but eventually this may lead to a genetic definition of what it is to be human. The amount of archaic DNA found in modern humans varies across the genome. This implies that negative selection may have been acting to remove archaic DNA. Regions that are thought to be functionally important appear to have less Neanderthal DNA. This is illustrated by the X chromosome, which has about five-fold less Neanderthal DNA than is found in autosomes. Further, testisspecific genes are found in regions that lack Neanderthal DNA,

perhaps indicating some level of male sterility in hybrids. There is also evidence for positive selection for Neanderthal sequences containing genes involved in skin and hair pigmentation.

plant history was in the gene families required for colonizing land, a completely new environment.

Genomes evolve at different rates

About one-third of the genes in Arabidopsis and rice appear to be plant-specific, that is, genes not found in any animal or fungal genome sequenced so far. These include the many thousands of genes involved in photosynthesis and photosynthetic anatomy. Among the remaining genes found in plants, many are very similar to those found in animal and fungal genomes, particularly the genes involved in basic intermediary metabolism, in genome replication and repair, and in transcription and protein synthesis. Prior to the availability of whole-genome sequences, assessment of the extent of genetic similarity and difference among diverse organisms had been difficult at best.

As noted earlier in this section, the genomes of viruses and bacteria can evolve in a matter of days. Evolution of insect genomes occurs more rapidly than that of mammalian genomes, which evolve over millions of years. At least a part of this is due to differences in generation time. However, even in some complex eukaryotic organisms, genome evolution can occur more rapidly. A comparison using a selection of plant and animal genomes yielded a surprising finding. Plant genomes change much more rapidly than animal genomes. This is especially evident in noncoding DNA, which tends to change more rapidly than protein-coding DNA. Many conserved, noncoding regions of DNA can be identified when a fish and a primate genome are aligned, even though the two diverged 400 mya. But very few similarities are found between the noncoding DNA of Arabidopsis and rice, Oryza sativa, although the two diverged only 200 mya. The sequencing of the corn genome has revealed that two varieties can differ by as much as 20%. Contrast this with the differences between human and chimp genomes. What accounts for all the variation in plant genomes? Transposable elements and other mobile elements frequently remodel the genome. Different bits of different genes come together in new combinations, and as a result, new regulatory elements are created.

Plant, fungal, and animal genomes have unique and shared genes We now step back farther and consider genomic differences among the eukaryotic kingdoms that diverged long before the examples just discussed. You have already seen that many genes are highly conserved in animals. Plant genes are also highly conserved, but new families of genes have emerged at each stage of plant evolution.

Evolution of plant-specific genes Sequenced plant genomes offer a window into a billion years of evolution. A total of 3814 gene families are shared by every plant, from the algae to grapes and corn. All the essentials for photosynthesis are found in the genome of the single-celled alga, Chlamydomonas reinhardtii, along with a gene that is now used to make flowers in the flowering plants. Although multicellularity was a huge evolutionary step, the multicellular alga Volvox has just about the same number of genes as Chlamydomonas. Lots of extra genes were not required for this major innovation. The move onto land is represented by the genome of the moss Physcomitrella patens that arose 450 mya. New genes that allowed the plant to survive in an arid environment arose, resulting in 3006 new gene families. Although moss are very different from flowering plants, making spores instead of seed, for example, they share 80% of the toolkit of developmental genes found in the flowering plant Arabidoposis. The shift from mosses to plants with internal transport systems required another 516 gene families. The transition to flowering plants, which now dominate land-based flora, required another 1350 gene families. It is perhaps not surprising that the biggest genomic change in the billion years of

Comparison of plants with animals and fungi

Learning Outcomes Review 24.1

Genomes vary in number of similar genes, arrangement of genes, total number of base-pairs, and base-pair differences. Closely related animals such as humans and chimps exhibit highly similar genomes, but greater variation is found in related plant genomes. ■■

Would you expect a high degree of similarity between genomes of a bony fish, such as a swordfish, and a cartilaginous fish, such as a shark? What genes might be different?

24.2 Genome

Size

Learning Outcomes 1. Differentiate between autopolyploidy and allopolyploidy. 2. Explain why most crosses between two species do not result in a new polyploid species. 3. Explain why the genome of a polyploid is not identical to the sum of the two parental genomes. 4. Explain why genome size and gene number do not correlate.

Both genome size and gene number vary greatly among the eukaryote species (see figure 24.1). One contributing factor is wholegenome duplication, which results in polyploidy, the focus of this section. Genome duplication alone, however, cannot explain why rice has about 40,000 genes distributed among 370 Mb of DNA, while within the about 3400 Mb of the human genome, only about 20,000 genes are found. To more fully understand the relationship between gene number and genome size in eukaryotes, the noncoding DNA must also be considered.

Data analysis Estimate and compare the number of genes per mega base-pair of genomic DNA in humans and rice. Which genomes in figure 24.1 are most like humans in terms of the ratio of genes to base-pairs?

chapter

24 Genome Evolution 509

Nicotiana sylvestris

Nicotiana tomentosiformis

Diploid (SS)

Diploid (TT)

ST hybrid

Figure 24.3 Allopolyploidy occurred in tobacco 5 mya, but can be approximated by crossing the progenitor species and initiating a doubling of chromosomes, often through tissue culture followed by plant regeneration, which can lead to chromosome doubling. Tobacco species have many chromosomes, not all of which are visible in a single plane of a cell.

Genome duplication

Nicotiana tabacum

Ancient and newly created polyploids guide studies of genome evolution Polyploidy (three or more chromosome sets) can give rise to new species, as you learned in chapter 22. Autopolyploids arise by genome duplication within a single lineage, and allopolyploids arise by hybridization of two lineages followed by genome duplication (figure 24.3). Genome duplication is more prevalent in plants than in animals, although animal polyploids are known, especially in fish and amphibians. In fact, the vertebrate lineage is thought to have undergone two ancient whole-genome duplication events. Two avenues of research have led to intriguing insights into genome alterations following polyploidization. The first approach studies ancient polyploids, called paleopolyploids. Sequence comparisons and phylogenetic tools establish the time and patterns of polyploidy events (figure 24.4). Sequence divergence between homologues, as well as the presence or absence of duplicated gene pairs from the hybridization, can be used for historical reconstructions of genome evolution. All copies of duplicate gene pairs arising through polyploidy are not necessarily found thousands or millions of years after polyploidization. The loss of duplicate genes is considered later in this section. The second approach is to create synthetic polyploids by crossing plants most closely related to the ancestral species and then chemically inducing chromosome doubling. Unless the hybrid genome becomes doubled, the plant will be sterile because it will lack homologous chromosomes that need to pair during metaphase I of meiosis. Because meiosis requires an even number of chromosome sets, species with ploidy levels that are multiples of two can reproduce sexually. However, meiosis is problematic in a 3n organism, 510 part IV Evolution

Average number of gene pairs

Allopolyploid (SSTT)

2

genome duplication loss of duplicate genes

1 0

75

50

25

0

Time (MYA)

Figure 24.4 Sequence comparisons of numerous genes in a polyploid genome tell us how long ago allopolyploidy or autopolyploidy events occurred. Complex

analyses of sequence divergence among duplicate gene pairs and presence or absence of duplicate gene pairs provide information about when both genome duplication and gene loss occurred. The graph reveals multiple polyploidy events over evolutionary time.

such as asexually propagated commercial bananas, since three sets of chromosomes can’t be evenly divided between two cells. Triploid bananas are seedless. The aborted ovules appear as the little brown dots in the center of any cross section of a banana.

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Inquiry question Sketch out what would happen in meiosis in a 3n banana cell, referring back to chapter 11 if necessary.

11–14

2.5– 4.5

Brassica

Arabidopsis thaliana

1–2

3–5

11

Gossypium (cotton)

Medicago truncatula

Glycine (soybean)

Lactuca (lettuce)

Helianthus (sunflower)

Solanum (potato)

Lycopersicon (tomato)

Hordeum (barley)

Triticum (wheat)

Oryza (rice)

Zea (maize)

Saccharum (sugarcane)

Sorghum

Figure 24.5  Polyploidy has occurred numerous times in the evolution of the flowering plants (the numbers indicate millions of years ago—mya).

13–15 25–40

50–70 150–170 Inferred polyploidy event (MYA)

While polyploidy is much more frequent in plants, it is not limited to the plant kingdom. For example, triploid populations of whiptail Cnemidophorus tesselatus) are found in southeastern lizards (­ Colorado. These lizards are all female, and they reproduce parthenogenetically (see chapter 51). That is, the triploid eggs are able to produce identical triploid, female offspring through mitosis, with no meiosis or fertilization needed for reproduction.

225–300

M. truncatula

Soybean

Genome size: 315 Mb

Genome size: 974 Mb

Evidence of ancient polyploidy is found in plant genomes Polyploidy has occurred numerous times in the evolution of the flowering plants (figure 24.5). The legume clade that includes the soybean (Glycine max), the plant Medicago truncatula (a forage legume often used in research), and the garden pea (Pisum sativum) underwent a major polyploidization event 44 to 58 mya and again 15 mya. A quick comparison of the genomes of soybean and M. truncatula reveal a huge difference in genome size (figure 24.6). In addition to increasing genome size through polyploidization, genomes like M. truncatula definitely downsized over evolutionary time as well. The total size of a genome cannot be explained solely on the basis of polyploidization. A number of independent whole-genome duplications in plants cluster around 65 mya, coinciding with a mass extinction event caused by a catastrophic change due to an asteroid impact or increased volcanic activity. Polyploids may have had a better chance of surviving the extinction event with increased genes and alleles available for selection.

Polyploidy induces elimination of duplicated genes Formation of an allopolyploid from two different species is often followed by a rapid loss of genes (figure 24.7) and rearrangement or loss of chromosomes. In some polyploids, however, loss of one copy of many duplicated genes arises over a much longer period.

15

44–58

Polyploidy event (MYA)

Figure 24.6 Genome downsizing. Genome downsizing must have occurred in Medicago truncatula (the numbers indicate millions of years ago—mya). In some species a great deal of gene loss occurs in the first few generations after polyploidization. Modern tobacco, Nicotiana tabacum, arose from the hybridization and genome duplication of a cross between Nicotiana sylvestris (female parent) and N. tomentosiformis (male parent). Researchers have reconstructed these events and followed chromosome loss in this synthetic N. tabacum. Curiously, the loss of chromosomes is not even. More N. tomentosiformis chromosomes are lost than those of N. sylvestris. Similar unequal chromosome loss has been observed in synthetic wheat hybrids. This pattern has also been observed in the closely related Arabidopsis thaliana and Arabidopsis lyrata, where A. chapter

24 Genome Evolution 511

Polyploidy can alter gene expression

SCIENTIFIC THINKING Hypothesis: Some duplicated genes may be eliminated after polyploidy. Prediction: More duplicate genes will be present when an allopolyploid forms than a few generations later. Test: Make a synthetic polyploid from two Nicotiana species and look at the chromosomes under a microscope then and after three generations of self-fertilizing offspring. Nicotiana sylvestris

Nicotiana tomentosiformis

Diploid

Diploid

Polyploidy Nicotiana tabacum Allopolyploid

Duplicate gene loss Nicotiana tabacum

Result: Over time, N. tomentosiformis chromosomes are lost or shortened. Conclusion: Chromosomes and genes are preferentially eliminated following polyploidy. Further Experiments: Why might the chromosomes and genes of one species be preferentially eliminated? How could you test your explanation?

Figure 24.7 Polyploidy may be followed by the unequal loss of duplicate genes from the combined genomes. lyrata has 500 more genes than A. thaliana. In the 10,000 years since the two species diverged, hundreds of thousands of small regions of the A. thaliana genome have been deleted. The differential loss may be due to different rates of genome replication, as is true for synthetic human–mouse hybrid cultured cells.

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Inquiry question Why does the number of duplicate genes decrease after multiple rounds of polyploidization?

512 part IV Evolution

A striking discovery is the change in gene expression that occurs in the early generations after polyploidization. Some of this may be connected to an increase in the methylation of cytosines in the DNA. Methylated genes are usually not transcribed, as described in chapter 16. Simply put, polyploidization can lead to a short-term silencing of some genes. In subsequent generations, methylation decreases.

Transposons are mobilized by polyploidization Barbara McClintock, who won the Nobel Prize for her discovery and characterization of transposable elements, hypothesized that transposons could be mobilized by what she called genome shock. There are some data on transposon activity following hybridization that support this hypothesis. During the early generations following a polyploidization event, transposition events appear to increase. Gene mutations arise if a new insertion is in the coding region of a gene. Insertions in promoter and other regulatory regions can change gene expression. Transposition can also result in chromosomal rearrangements when a portion of the genome is relocated. All of these changes increase genetic variation, potentially leading to rapid evolution. The relative number of transposable elements in a species may influence the rate of genome evolution whether or not hybridization occurs. The orangutan’s genome has evolved more slowly than either of its close relatives—humans and chimps. Comparisons of the recently sequenced orangutan genome with chimp and humans revealed many fewer transposable elements in the orangutan.

Polyploidy alone cannot account for variation in genome size It is difficult to account for all genome size variation with polyploidy. Even with gene or chromosome elimination following hybridization, polyploidy is unlikely to result in such a range of ratios of gene number to genome size. Humans have nine times the amount of DNA found in the 3.93 × 108-bp pufferfish genome, but about the same number of genes. Plants have an even greater range of genome sizes. As much as a 200-fold difference has been found, yet all these plants weigh in with about 24,000 to 60,000 genes. Tulips, for example, have 170 times more DNA than Arabidopsis.

Noncoding DNA inflates genome size Why do humans have so much extra DNA compared with pufferfish? Much of it appears to be in the form of introns, noncoding segments (ncDNA) within a gene’s sequence, that are substantially bigger than those in pufferfish. The Fugu genome has only a handful of “giant” genes containing long introns; studying them should provide insight into the evolutionary forces that have driven the change in genome size during vertebrate evolution. Large expanses of retrotransposon DNA contribute to the differences in genome size from one species to another. Although part of the genome, ncDNA does not contain genes in the usual sense. As another example, Drosophila exhibits less ncDNA than

Anopheles, although the evolutionary force driving this reduction in noncoding regions is unclear. The 370-Mb rice genome and the 2-Gb maize genome are now fully sequenced. Both have about 40,000 protein-coding genes, roughly twice the number of genes in the human genome. Maize contains lots of repetitive DNA, which has increased its DNA content, but not its gene content. Comparisons between the rice, maize, and wheat genomes should provide clues about the genome of their common ancestor and the dynamic evolutionary balance between opposing forces that increase genome size (polyploidy, transposable element proliferation, and gene duplication) and those that decrease genome size (mutational loss).

Learning Outcomes Review 24.2

Autopolyploids arise from duplication of a species’ genome; allopolyploids occur from hybridization between two species. Unless the number of chromosomes doubles, an allopolyploid will likely be sterile because of lack of homologous pairs at meiosis. Polyploidization can lead to major changes in genome structure, including loss of genes, alteration of gene expression, increased transposon hopping, and chromosomal rearrangements. Polyploidy is considered important in the generation of biodiversity and adaptation, especially in plants, but it cannot explain why increases or decreases in genome size do not correlate with the number of genes. Evidently DNA content is not the same as gene content. Polyploidy in plants does not by itself explain differences in genome size. Often a greater amount of DNA is explained by the presence of introns and non-proteincoding sequences than by gene duplicates. ■■

How might a genome with a small number of genes and a small number of total base-pairs evolve into a genome with the same small number of genes and a thousand-fold larger genome?

24.3 Evolution

Within Genomes

Learning Outcomes 1. Define the terms segmental duplication, genome rearrangement, and pseudogene. 2. Explain why horizontal gene transfer can complicate evolutionary hypotheses.

Genome evolution can occur at the level of whole-genome duplication, but also occurs within genomes. Alterations are seen at all levels from individual genes to chromosomal rearrangements.

Individual chromosomes may be duplicated Aneuploidy refers to the gain or loss of an individual chromosome rather than of an entire genome. Failure of a pair of homologous chromosomes or sister chromatids to separate during meiosis is the most common way that aneuploidy occurs. In general, plants are

better able to tolerate aneuploidy than animals, but the explanation for this difference is elusive.

DNA segments may be duplicated One of the greatest sources of novel traits in genomes is duplication of segments of DNA. Two genes within an organism that have arisen from the duplication of a single gene in an ancestor are called paralogs. In contrast, orthologs reflect the inheritance of a single gene from a common ancestor—a conserved gene. When a gene is duplicated, the most likely fates of the duplicate gene are (1) losing function through subsequent mutation (becomes a pseudogene), (2) gaining a novel function through subsequent mutation (neofunctionalization), and (3) having the total function of the ancestral gene partitioned into the two duplicates (subfunctionalization). Gene families grow through gene duplication. In reality, however, most duplicate genes lose function. Why, then, do we think that gene duplication is a major evolutionary force for genes to gain new function? Part of the answer lies in where gene duplication is most likely to occur in the genome. In humans, the highest rates of duplication lie in the three most gene-rich chromosomes. Conversely, the seven chromosomes with the fewest genes show the least amount of duplication. Note that gene-poor does not necessarily mean less total DNA. Even more compelling, certain types of genes appear more likely to be duplicated: growth and development genes, immune system genes, and cell-surface receptors. Finally, and importantly, gene duplication is thought to be a major evolutionary force for gene innovation because the duplicated genes are found to have different patterns of gene expression. For example, the two duplicated copies may be expressed in different or overlapping sets of tissues or organs during development. About 5% of the human genome consists of segmental duplications, which are duplications of blocks of genes (figure 24.8). Segmental duplications may account for differences between human and chimp. Species-specific segmental duplications tend to contain genes that are differentially expressed between the species. A simple explanation is that there are more copies of the gene being expressed, but experimental evidence shows that it is more likely that the regulatory DNA near the duplicate is different. Both rice and Arabidopsis have higher copy numbers for gene families (multiple slightly divergent copies of a gene) than are seen in animals or fungi, suggesting that these plants may have undergone numerous episodes of segmental duplication during the 150 to 200 million years since rice and Arabidopsis diverged from a common ancestor. As whole-genome duplications have also occurred since they diverged, additional analysis will be needed to determine the relative effects of polyploidy and segmental duplication in plant evolution.

Genomes may become rearranged Humans have one fewer chromosome than chimpanzees, gorillas, and orangutans (figure 24.9). It’s not that we have lost a chromosome. Rather, at some point in time, two midsized ape chromosomes fused to make what is now human chromosome 2, the secondlargest chromosome in our genome. chapter

24 Genome Evolution 513

Figure 24.8 Segmental duplication on the human Y chromosome. Each orange region has 98% sequence similarity with a sequence on a different human chromosome. Each dark blue region has 98% sequence similarity with a sequence elsewhere on the Y chromosome. Figure 24.9 Living great apes. All living great apes,

with the exception of humans, have a haploid chromosome number of 24. Humans have not lost a chromosome; rather, two smaller chromosomes fused to make a single chromosome.

Orangutan

Gorilla

Chimpanzee

24 chromosomes

24 chromosomes

24 chromosomes

Hominid

23 chromosomes

Apes

The fusion leading to human chromosome 2 is an example of the sort of genome reorganization that has happened in the vertebrate lineage since the 54-chromosome protovertebrate genome described earlier. The remains of some of these rearrangements are visible in the structural variation observed between species. Chromosome rearrangements are common, yet over long segments of chromosomes, the linear order of mouse and human genes is the same—the common ancestral sequence has been preserved in both species. This conservation of synteny (see chapter 18) was anticipated from earlier gene mapping studies, and it provides strong evidence that evolution actively shapes the organization of the eukaryotic genome. As seen in figure 24.10, the conservation of synteny allows researchers to more readily locate a gene in a different species using information about synteny, thus underscoring the power of a comparative genomic approach.

1500 OR genes—about 40% more OR genes than humans. Only 20% of the mouse OR genes are pseudogenes, compared to 63% in humans, which are inactive pseudogenes (genes that do not produce a functional product due to premature stop codons, missense mutations, or deletions). In contrast, half the chimpanzee and gorilla OR genes function effectively, and over 95% of New World monkey OR genes appear to be functional. The most likely explanation for these differences is that humans came to rely on other senses, reducing the selection pressure against loss of OR gene function by random mutation. An older question about the possibility of positive selection for OR genes in chimps was resolved with the completion of the chimp genome. A careful analysis indicated that both humans and chimps are gradually losing OR genes to pseudogenes and that there is no evidence to support positive selection for any of the OR genes in the chimp.

Gene inactivation results in pseudogenes

Rearranged DNA can acquire new functions

The loss of gene function is another important way genomes evolve. Consider the olfactory receptor (OR) genes that are responsible for our sense of smell. These genes code for receptors that bind odorants, initiating a cascade of signaling events that eventually lead to our perception of scents. Gene inactivation seems to be the best explanation for our reduced sense of smell relative to that of the great apes and other mammals. Mice have the largest mammalian OR gene family, with about

Errors in meiosis that rearrange parts of genes most often create pseudogenes, but occasionally a broken piece of a gene can end up in a new spot in the genome where it acquires a new function. One of the most intriguing examples occurred within a family of fish in the suborder Notothenioidae found in the Antarctic Ocean. These fish are called icefish because they survive the frigid temperatures in the Antarctic, in part because of a protein in their blood that works like antifreeze. Reconstructing evolutionary history using

514 part IV Evolution

Interchromosomal duplications Intrachromosomal duplications Not sequenced Heterochromatin that is not expressed

Soybean d2

K

2

c2

b2

c1

3

M. truncatula

Figure 24.10 Synteny and gene identification. Genes sequenced in the model legume, Medicago truncatula, can be used to identify homologous genes in soybean, Glycine max, because large regions of the genomes are syntenic, as illustrated for some of the linkage groups (chromosomes) of the two species. Regions of the same color represent homologous genes. comparative genomics reveals that a 9-bp fragment of a gene coding for a digestive enzyme moved to a new location where it evolved to encode part of an antifreeze protein. The series of errors that gave rise to the new protein persisted only because the change coincided with a massive cooling of the Antarctic waters. Natural selection acted on this mutation over millions of years.

species seem to have been less firm than they are now and DNA more readily moved among different organisms. Earlier in the history of life, gene swapping between species was rampant. Today HGT continues in prokaryotes and eukaryotes, but less frequently. An intriguing example of more recent HGT between moss and a flowering plant is described in chapter 30.

Noncoding DNA can acquire regulatory functions

Gene swapping in early lineages

So far, we have primarily compared genes that code for proteins. As more genomes are sequenced, we learn that much of the genome is composed of ncDNA. The repetitive DNA is often retrotransposon DNA, contributing to as much as 30% of animal genomes and 40 to 80% of plant genomes. (Refer to chapter 18 for more information on repetitive DNA in genomes.) Yet there are conserved noncoding regions (CNCs) that evolved much more slowly than expected, assuming there was no associated function. An analysis of CNCs in 40 vertebrates led to the conclusion that as the rate of base-pair substitution slowed down over evolutionary time, these CNCs had begun to regulate the expression of transcription factors, genes that specifically influence development, and genes that encode receptor-binding proteins that are important in signaling.

Horizontal gene transfer complicates matters Evolutionary biologists build phylogenies on the assumption that genes are passed from generation to generation, a process called vertical gene transfer (VGT). Hitchhiking genes from other species, a process referred to as horizontal gene t­ ransfer (HGT) and sometimes called lateral gene transfer, can lead to phylogenetic complexity. HGT was likely most prevalent very early in the history of life, when the boundaries between individual cells and

The extensive gene swapping among early organisms has caused many researchers to reexamine the base of the tree of life. Early phylogenies based on ribosomal RNA (rRNA) sequences indicate that an early prokaryote gave rise to two major domains: the Bacteria and the Archaea. From one of these lineages, the domain Eukarya emerged; its organelles originated as unicellular organisms engulfed specialized prokaryotes. This rRNA phylogeny is being revised as more microbial genomes are sequenced. In 2015, the National Center for Biotechnology Information (NCBI) databases contained 33,543 prokaryotic genomes. With new sequencing technology, microbial genomes can be sequenced in less than a day. Phylogenies built with rRNA sequences suggest that the domain Archaea is more closely related to the Eukarya than to the Bacteria. But as more microbial genomes are sequenced, investigators find bacterial and archaeal genes showing up in the same organism! The most likely conclusion is that organisms swapped genes, even absorbing DNA obtained from a food source. Perhaps the base of the tree of life is better viewed as a web than as a branch (figure 24.11).

Evidence for gene swapping in the human genome Let’s move closer to home and look at the human genome, which is riddled with foreign DNA, often in the form of transposons. The many transposons of the human genome provide a paleontological record over several hundred million years. chapter

24 Genome Evolution 515

Bacteria

Archaea

Eukarya

24.4 Gene

Function and Expression Patterns

Learning Outcomes 1. Explain how species with nearly identical genes can look very different. 2. Describe the action of the FOXP2 gene across species.

C

rop hlo

s last

to Mi

o ch

ria nd

Figure 24.11 Horizontal gene transfer. Early in the history

of life, organisms may have freely exchanged genes beyond multiple endosymbiotic events. To a lesser extent, this transfer continues today. The tree of life may be more like a web or a net.

Comparisons of versions of a transposon that has duplicated many times allow researchers to construct a “family tree” to identify the ancestral form of the transposon. The percentage of sequence divergence found in duplicates allows an estimate of the time at which that particular transposon originally invaded the human genome. In humans, most of the DNA hitchhiking seems to have occurred millions of years ago in very distant ancestor genomes. Our genome carries many more ancient transposons than do the genomes of Drosophila, C. elegans, and Arabidopsis. One explanation for the observed low level of transposons in Drosophila is that fruit flies somehow eliminate unnecessary DNA from their genome 75 times faster than humans do. Our genome has simply hung on to hitchhiking DNA more often. The human genome has had minimal transposon activity in the past 50 million years; mice, by contrast, are continuing to acquire new transposable elements. This difference may explain in part the more rapid change in chromosome organization in mice than in humans.

Learning Outcomes Review 24.3

In segmental duplication, part of a chromosome and the genes it contains are duplicated. In genome rearrangement, segments of chromosomes may change places or chromosomes may fuse with one another. Pseudogenes have become inactivated in the course of evolution but still persist in the genome. All these changes have evolutionary consequences. Horizontal gene transfer has led to an unexpected mixing of genes among organisms, creating many phylogenetic questions. ■■

How would you determine whether a gene was a pseudogene or an example of horizontal gene transfer?

516 part IV Evolution

Gene function can be inferred by comparing genes in different species. When the human genome was first compared to another mammalian genome, the mouse, 1000 genes with unknown function had a function assigned. One of the major puzzles arising from comparative genomics is that organisms with very different forms can share so many conserved genes in their genomic toolkit. The best explanation for why a mouse develops into a mouse and not a human is that the same or similar genes are expressed at different times, in different tissues, and in different amounts and combinations. For example, the cystic fibrosis gene (cystic fibrosis transmembrane conductance regulator, CFTR), which has been identified in both species and affects a chloride ion channel, illustrates this point. Defects in the human CFTR gene cause especially devastating effects in the lungs, but mice with the mutant CFTR gene do not have lung symptoms. Possibly variations in expression of CFTR between mouse and human explain the difference in lung symptoms when CFTR is defective.

Chimp and human gene transcription patterns differ One approach to analyzing transcription patterns in humans and chimps has been to use RNA sequencing (RNA-seq; see chapter 18) to measure mRNA from single-cells taken from cerebral organoids. Cerebral organoids are cultured from pluripotent stem cells that are induced to differentiate into organized neural tissue that include radial glial cells and excitatory neurons. Comparing chimp and human cerebral organoids, investigators found 383 genes up-regulated in radial glial cells and 220 genes up-regulated in excitatory neurons. Conversely, 285 genes in radial glial cells, and 165 genes in excitatory neurons, were down-regulated. A subset of these genes proved to be associated with human-specific structural variations. While this currently falls into the category of intriguing possibilities, it highlights one approach to finding functional significance in genomic differences.

The FOXP2 gene is involved in speech Language, conveyed by complex vocalizations, is one of the defining characteristics of humans. This has inspired generations of geneticists to search for genes involved in all aspects of language skills. While the use and acquisition of language show high heritability, the search for the source of this variation has produced mixed results. One clear exception is the FOXP2 gene, which encodes a transcription factor expressed in the brain. Individuals heterozygous

for a mutant allele of FOXP2 show difficulties in learning and sequencing the facial movements necessary to produce speech, but not impaired language comprehension. The function of FOXP2 is necessary for the development of brain circuits in the neocortex, basal ganglia, and cerebellum, which are involved in language and fine motor control. Analysis of the evolution of this gene has only added to interest in its role. In comparisons between humans and mice, FOXP2 is among the most conserved 5% of genes. Despite this conservation, the rate of amino acid substitution in FOXP2 protein in humans is increased 50-fold over the average rate in the genome. It is also found in a region of the genome that has little archaic DNA. There are three amino acid substitutions in the human protein compared to the protein in mice. Of these, two occurred after humans diverged from chimps and bonobos. The FOXP2 protein is identical in all nonhuman primates examined, with the exception of a single amino acid substitution in the orangutan (figure 24.12). The downstream targets of the FOXP2 transcription factor have not been characterized, but there are interesting hints that its role in communication may not be limited to humans. Song birds with the level of FOXP2 protein reduced by RNA interference show disturbed development of song variability, especially learning by imitation. Mice communicate via squeaks, with lost young mice emitting high-pitched squeaks. FOXP2 mutations leave mice squeakless and transgenic baby mice carrying the human FOXP2 squeak differently. Both downstream targets of FOXP2 protein and its role in other species are areas of active research that should shed further light on the role of FOXP2 in human evolution.

Chimp p

Mouse

H Human Gorilla

Rhesus monkey

0/5 2/0 Orangutan

0/2

0/2

0/7 0/5 1/131

1/2 0/2 Synonymous changes Nonsynonymous changes

Figure 24.12 Evolution of FOXP2. Beige bars represent

synonymous nucleotide substitutions and brown bars represent nonsynonymous changes. Numbers indicate nonsynonymous over synonymous nucleotide substitutions unique to each lineage. Humans have two amino acid substitutions not found in other primates.

Learning Outcomes Review 24.4

To understand functional differences between genes shared by species, one must look beyond sequence similarity. Alterations in the time and place of gene expression can lead to marked differences in phenotype. As an example, the FOXP2 gene appears to be involved in sound production in mice, chimps, gorillas, macaques, humans, and songbirds, and only very small differences may have led to human speech. ■■

How can a single-nucleotide difference in a gene lead to a noticeably different phenotype? Give examples.

24.5 Applying

Comparative Genomics

Learning Outcomes 1. Describe how comparative genomics can reveal the genetic basis for disease. 2. Explain how genome comparisons between a pathogen and its host can aid drug development. 3. Describe how genome comparisons can be useful when working with endangered species.

Comparisons among individual human genomes continue to provide information on genetic disease detection and the best course of treatment. An even broader array of possibilities arises when comparisons are made among species. There are advantages to comparing both closely and distantly related pairs of species, as well as comparing the genomes of a pathogen and its host. Genomelevel comparisons are also assisting conservation biologists in developing breeding programs. Examples of the benefits of each type of genome comparison follow.

Distantly related genomes offer clues for causes of disease Sequences that are conserved between humans and pufferfish provide valuable clues for understanding the genetic basis of many human diseases. Amino acids critical to protein function tend to be preserved over the course of evolution, and changes at such sites within genes are more likely to cause disease. It is difficult to distinguish functionally conserved sites when comparing human proteins with those of other mammals because not enough time has elapsed for sufficient changes to accumulate at nonconserved sites. A promising exception is the duck-billed platypus (Ornithorhynchus anatinus), which diverged from other mammals about 166 mya and whose genome provides clues to the evolution of the immune system. Because the pufferfish genome is only distantly related to the human genome, conserved sequences are far more easily distinguished than even in the platypus. chapter

24 Genome Evolution 517

Closely related organisms enhance medical research Because studies in humans must be strictly regulated for ethical reasons, it is much easier to design experiments to identify gene function in an experimental system like that of the mouse. Comparing mouse and human genomes quickly revealed the function of 1000 previously unidentified human genes. The effects of these genes can be studied in mice, and the results can be used in potential treatments for human diseases. A draft of the rat genome has been completed, and even more exciting news about the evolution of mammalian genomes may emerge from comparisons of these species. One of the most exciting aspects of comparing rat and mice genomes is the potential to capitalize on the extensive research on rat physiology, especially heart disease, and the long history of genetics in mice. Linking genes to disease has become much easier. As described in section 24.3, the human genome contains segmental duplications that are absent in the chimp genome. Some of these duplications contain genes with alleles that produce human disease. For example, some of the regions duplicated only in humans correspond to regions implicated in Prader–Willi syndrome and spinal muscular atrophy. The significance of these observations is not clear, but there is great interest in analyzing regions that differ between humans and closely related species.

Pathogen–host genome differences reveal drug targets With genome sequences in hand, pharmaceutical researchers are more likely to find suitable drug targets to eliminate pathogens without harming the host. Diseases—including malaria and Chagas disease—in many developing countries have both human and insect hosts. Both of these infections are caused by protists (see chapter 28), and the value of comparative genomics in drug discovery to treat them is illustrated later in this section.

Malaria Anopheles gambiae, the malaria-carrying mosquito, along with Plasmodium falciparum, the protistan parasite it transmits, together have an enormous effect on human health, resulting in 1.7 to 2.5 million deaths each year from malaria. The genomes of both Anopheles and Plasmodium were sequenced in 2002. P. falciparum, which causes malaria, has a relatively small genome of 2.33 × 107 bp that proved very difficult to sequence. It has an unusually high proportion of adenine and thymine, making it hard to distinguish one portion of the genome from the next. The project took five years to complete. P. falciparum appears to have about 5500 genes, with those of related function clustered together, suggesting that they might share the same regulatory DNA. P. falciparum is a particularly crafty organism that hides from our immune system inside red blood cells, regularly changing the proteins it presents on the surface of the red blood cell. This “cloaking” has made developing a vaccine or other treatment for malaria particularly difficult. Recently, a link to chloroplast-like structures in P. falciparum has raised other possibilities for treatment. An odd subcellular component called the apicoplast, found only in Plasmodium 518 part IV Evolution

2 μm

Figure 24.13 Plasmodium apicoplast. Drugs targeting enzymes used for fatty acid biosynthesis within Plasmodium apicoplasts (colored dark green) offer hope for treating malaria.

Courtesy of Dr. Lewis G. Tilney and Dr. David S. Roos, University of Pennsylvania

and its relatives, appears to be derived from a chloroplast appropriated from algae engulfed by the parasite’s ancestor (figure 24.13). Analysis of the Plasmodium genome reveals that about 12% of all the parasite’s proteins, encoded by the nuclear genome, head for the apicoplast. These proteins act there to produce fatty acids. The apicoplast is the only location in which the parasite makes the fatty acids, suggesting that drugs targeted at this biochemical pathway might be very effective against malaria. Another disease-prevention possibility is to look at chloroplastspecific herbicides, which might kill Plasmodium by targeting the chloroplast-derived apicoplast. Coupled with a newer vaccine, this treatment could substantially reduce the incidence of malaria.

Chagas disease Trypanosoma cruzi, an insect-borne protozoan, kills about 21,000 people in Central and South America each year. As many as 18 million suffer from this infection, called Chagas disease, the symptoms of which include damage to the heart and other internal organs. Genome sequencing of T. cruzi was completed in 2005. A surprising and hopeful finding is that a common core of 6200 genes is shared among T. cruzi and two other insect-borne pathogens: Trypanosoma brucei and Leishmania major. T. brucei causes African sleeping sickness, and L. major infections result in lesions of the skin of the limbs and face. These core genes are being considered as possible targets for drug treatments. Currently, no effective vaccines and only a few drugs with limited effectiveness are available to treat any of these diseases. The genomic similarities may not only aid in targeting drug development, but perhaps also result in a treatment or vaccine that is effective against all three devastating illnesses (figure 24.14).

Genome comparisons inform conservation biology Genomes of species on the brink of extinction are being mined for information that could contribute to disease reduction and con­ servation efforts. Here we consider the application of genomics

Figure 24.14 Comparative genomics may aid in drug development. The organisms that

Chagas disease

cause Chagas disease, African sleeping sickness, and leishmaniasis, which claim millions of lives in developing nations each year, share 6200 core genes. Drug development targeted at proteins encoded by the shared core genes could yield a single treatment for all three diseases. (left) Eye of Science/Science

African sleeping sickness

2 μm

Source; (middle) David Spears FRPS FRMS/ Corbis Documentary/Getty Images; (right) KATERYNA KON/Science Photo Library/Alamy Stock Photo

Leishmania infection

3 μm

5 μm

6200 shared core genes

Target for drug development

to three endangered species, the Tasmanian devil (Sarcophilus harrisii), the giant panda (Ailuropoda melanoleura), and the polar bear (Ursus maritimus). The Tasmanian devil, a marsupial on the Australian island of Tasmania, faces decimation from devil facial tumor disease (DFTD, figure 24.15). Close to 90% of the population is affected, and since 1996, 60% of the devils have been wiped out due to DFTD. A comparison of genomes from two devils, named Cedric and Spirit, from distant corners of Tasmania revealed extremely low genetic diversity that can be traced back 100 years before the DFTD outbreak. Only the now extinct Tasmanian tiger had less genetic diversity (­figure  24.16). Breeding programs can use the genomic information to preserve what diversity there is in the population. Sequencing of the genome of the giant panda offered more promising news about population diversity (figure 24.16). The panda has 2.7 million SNPs, which is three times as many as were identified in the Tasmanian devil. Bamboo is the mainstay of a

panda diet, and habitat destruction is a factor in the decline of pandas. Curiously, the panda’s relatives are carnivores, and pandas maintain the genes associated with carnivores. Furthermore, they lack the genes coding for enzymes needed to fully digest bamboo. Attention is now focused on the microbes in the panda’s gut that digest bamboo. Collectively, this information may assist conservationists in keeping the population from dipping below its current level of 2500 to 3000 individuals. An unexpected finding arose when the mitochondrial genomes of modern and fossil polar and brown bears (including DNA from the jaw of a 110,000- to 130,000-year-old polar bear) were sequenced. Polar bears evolved about 150,000 years ago. Geneticists were shocked to find that the entire maternal line of polar bears can be traced back to a brown bear living in Ireland between 20,000 and 50,000 years ago. Despite the close relation to brown bears and the ability to interbreed, this finding does not offer much hope for the polar bears facing extinction as warmer temperatures melt their sea ice homes. Average temperatures for the next 50  years are predicted to be much warmer than anything polar bears have experienced in their 150,000 years on Earth or the 20,000 or so years since they mated with brown bears.

Learning Outcomes Review 24.5

DNA sequences conserved over evolutionary time tend to be those critical for protein function and survival. Variations in these conserved sequences may provide clues to diseases with a hereditary component. Knowledge of a pathogenic organism’s genome and its differences from a host’s genome may allow targeting of drugs and vaccines that affect the invader but leave the host unharmed. Comparative genomics within species can provide information about population diversity of endangered species. Conservation biologists can use this information to develop breeding strategies. ■■

Figure 24.15 DFTD is a fatal condition for Tasmanian devils. Tumors interfere with eating, and devils die within months of the first appearance of lesions. Dave Watts/Alamy Stock Photo

A pathogen makes a critical protein that differs from the human version by only seven amino acids. What approaches might lead to an effective drug against the pathogen? What drawbacks might be encountered?

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24 Genome Evolution 519

100 Endangered Extinct Not endangered

92 85

80

77

70

67

60 50 43 8 28

20

25

25

20

19

Stellar sea lion

European human

Brown bear

Mammoth clade I

Whale

Mammoth clade II

Polar bear

Panda

Wolf

Bushman of southern Africa

0

Gorilla

10

15

10

5 Tasmanian tiger

30

Tasmanian devil

44

40

Bison

Genetic diversity within species

90

Figure 24.16 Genetic diversity of mitochondrial genomes based on average number of differences between pairs of individuals in the populations indicated.

Chapter Review Stephen Robinson/NHPA/Photoshot/Newscom

24.1 Comparative Genomics

Evolution of the vertebrate genome. The human genome has been compared to many other vertebrates. This has led to identification of vertebrate-specific genes. This can be taken farther to hypothesize a protovertebrate karyotype of 54 chromosomes.

Plant, fungal, and animal genomes have unique and shared genes. Plant genomes have changed much more rapidly than animal genomes. It appears that about one-third of plant genes are unique to plants. Of the remaining plant genes, many are also found in animal and fungal genomes and are required for metabolism and gene expression.

The human genome shares similarities with those of great apes. Human genomes are very similar to the genomes of great apes. Primate genomes share the feature of a large amount of repetitive DNA, including transposable elements. These transposons show speciesspecific expansion and deletion. These comparisons are beginning to find possible functional differences.

24.2 Genome Size

The human genome contains sequences from extinct primates. The human genome contains sequences that are the result of ancient hybridization events with Neanderthal and Denisovans, two extinct primates. Analyzing these differences shed light on human evolution.

Evidence of ancient polyploidy is found in plant genomes. Polyploidy has occurred numerous times in the evolution of flowering plants, and downsizing of genomes is common.

Genomes evolve at different rates. Viral, bacterial, and even insect genomes evolve more rapidly than mammalian genomes. Plant genomes change more quickly than animal genomes, possibly as the result of massive genome remodeling from extensive transposition of mobile elements. 520 part IV Evolution

Ancient and newly created polyploids guide studies of genome evolution. Autopolyploidy results from an error in meiosis that leads to a duplicated genome; allopolyploidy is the result of hybridization between species (figure 24.3).

Polyploidy induces elimination of duplicated genes. Downsizing of a polyploid genome can be caused by unequal loss of duplicated genes (figure 24.7). Polyploidy can alter gene expression. Polyploidization can lead to short-term silencing of genes via methylation of cytosines in the DNA.

Transposons are mobilized by polyploidization. Transposons become highly active after polyploidization; their insertions into new positions may lead to new phenotypes. Polyploidy alone cannot account for variation in genome size. The vast range of ratios of gene number to genome size, even in closely related species, cannot be explained by polyploidy alone. Noncoding DNA inflates genome size. Genome size is most often inflated due to the presence of introns and non-protein-coding sequences. Genome size does not correlate with the number of genes. Some species have very little noncoding DNA, and others have extensive amounts of ncDNA, often retrotransposon DNA. Two species, such as rice and maize, can vary substantially in the amount of ncDNA and yet have similar numbers of genes.

24.3 Evolution Within Genomes Individual chromosomes may be duplicated. Aneuploidy, the duplication or loss of individual chromosomes, results from errors in meiosis. It is tolerated better in plants than in animals. DNA segments may be duplicated (figure 24.8). Paralogs are duplicated ancestral genes; orthologs are conserved ancestral genes. Duplicated DNA is common for genes associated with growth and development, immunity, and cell-surface receptors. Genomes may become rearranged. Genomes may be rearranged by moving gene locations within a chromosome or by the fusion of two chromosomes. Conservation of synteny refers to preservation of long segments of ancestral chromosome sequences identifiable in related species (figure 24.10). Gene inactivation results in pseudogenes. Some ancestral genes become inactivated as they acquire mutations and are termed pseudogenes. Rearranged DNA can acquire new functions. Occasionally part of a gene can end up in a new spot in the genome where its function changes.

Noncoding DNA can acquire regulatory functions. DNA that has no function in an organism can acquire mutations without any detrimental effect. However, conserved noncoding regions (CNCs) evolved more slowly than expected. These CNC regions appear to have gained functions that regulate the expression of neighboring proteincoding genes. Horizontal gene transfer complicates matters. Horizontal gene transfer creates many phylogenetic questions, such as the origins of the three major domains (figure 24.11).

24.4 Gene Function and Expression Patterns Chimp and human gene transcription patterns differ. Even when species have highly similar genes, expression of these genes may vary greatly. Posttranscriptional differences may also contribute to species differences. The FOXP2 gene is involved in speech. Small evolutionary changes in the FOXP2 protein and its expression may have influenced the evolution of human speech (figure 24.12).

24.5 Applying Comparative Genomics Distantly related genomes offer clues for causes of disease. Changes in amino acid sequences of critical proteins are a likely cause of diseases, and these differences can be identified by genome comparison. Closely related organisms enhance medical research. By comparing related organisms, researchers can focus on genes that cause diseases and devise possible treatments. Pathogen–host genome differences reveal drug targets. Analysis of the genomes of pathogenic organisms may provide new avenues of treatment and prevention (figure 24.14). Genome comparisons inform conservation biology. Comparative genomics within species can provide information about population diversity of endangered species. Conservation biologists can use this information to develop breeding strategies.

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24 Genome Evolution 521

Visual Summary Genomic analysis includes

Comparative genomics

reveals

informs more variation

Phylogeny

Rate of evolution

less variation faster

Plants

Animal

affected by

Duplication

Transposable elements

Rearranged DNA

whole genome

and

and

Polyploidy

Noncoding DNA

Plants

identified orthologs Vertebrates

Loss of genes

often followed by

522 part IV Evolution

Polyploidy

Chromosome fusion

Loss of chromosomes

Segmental duplication

transposable elements Primates

Noncoding DNA

Aneuploidy

duplications, fusions, Rearrangements rearrangements Amniotes

acquiring new function

increases due to

slower Animal

Genome evolution

Genome size

Pseudogene

loss of function

Functional genomics

complicated by

Horizontal gene transfer can occur by

Transcription activity Gene expression differences in

examining Species-specific genes and

applications in

Species-specific expression changes in Medicine Timing Tissues Amounts

Conservation biology

Review Questions Stephen Robinson/NHPA/Photoshot/Newscom

U N D E R S TA N D 1. Humans and pufferfish diverged from a common ancestor about 450 mya, and these two genomes have a. b. c. d.

very few of the same genes in common. all the same genes. a large proportion of the genes in common. no nucleotide divergence.

2. Genome comparisons have suggested that mouse DNA has mutated about twice as fast as human DNA. What is a possible explanation for this discrepancy? a. Mice are much smaller than humans. b. Mice live in much less sanitary conditions than humans and are therefore exposed to a wider range of mutation-causing substances. c. Mice have a smaller genome size. d. Mice have a much shorter generation time. 3. Polyploidy in plants a. has only arisen once and therefore is very rare. b. only occurs naturally when there is a hybridization event between two species. c. is common, but never occurs in animals. d. is common, and does occur in some animals. 4. Homologous genes in distantly related organisms can often be easily located on chromosomes due to a. b. c. d.

horizontal gene transfer. conservation of synteny. gene inactivation. pseudogenes.

5. All of the following are believed to contribute to genomic diversity among various species, except a. b. c. d.

gene duplication. gene transcription. lateral gene transfer. chromosomal rearrangements.

6. What is the fate of most duplicated genes? a. b. c. d.

Gene inactivation Gain of a novel function through subsequent mutation They are transferred to a new organism using lateral gene transfer. They become orthologues.

A P P LY 1. Chimp and human DNA whole-genome sequences differ by about 1.23%. Determine which of the following explanations is most consistent with the substantial differences in morphology and behavior between the two species.

c. d.

It cannot be explained with current genetic theory. The differences are caused by random effects during development.

2. You are offered a summer research opportunity to investigate a region of ncDNA in maize. A friend politely smiles and says that only graduate students get to work on the coding regions of DNA. How would you critique your friend’s statement? a. b. c. d.

The friend has a point; ncDNA is “junk” DNA and therefore not very important. The ncDNA produces protein through mechanisms other than transcription. Most ncDNA is usually translated. Often ncDNA produces RNA transcripts that themselves have regulatory function.

3. Analyze the conclusion that the Medicago truncatula genome has been downsized relative to its ancestral legume, and circle the evidence that is consistent with this conclusion. a. b. c. d.

Medicago has a proportional decrease in the number of genes. Medicago has a proportional increase in the number of genes. Medicago has an increase in the amount of DNA. Medicago has a decrease in the amount of DNA.

4. Analyze why an herbicide that targets the chloroplast is effective against malaria. a. b. c. d.

Because Plasmodium needs a functional apicoplast Because the main vector for malaria is a plant Because mosquitoes require plant leaves for food Because Plasmodium mitochondria are very similar to chloroplasts

SYNTHESIZE 1. The FOXP2 gene is associated with speech in humans. It is also found in chimpanzees, gorillas, orangutans, rhesus macaques, and even the mouse, yet none of these mammals speak. Develop a hypothesis that explains why FOXP2 supports speech in humans but not other mammals. 2. One of the common misconceptions about sequencing projects (especially the high-profile Human Genome Project) is that creating a complete road map of the DNA will lead directly to cures for genetically based diseases. Given the percentage similarity in DNA between humans and chimps, is this simplistic view justified? Explain. 3. How does horizontal gene transfer (HGT) complicate phylogenetic analysis?

a. It must be due largely to gene expression. b. It must be due exclusively to environmental differences.

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24 Genome Evolution 523

Part V IIIDiversity Genetic and Molecular Part of Life on EarthBiology

25 CHAPTER

The Origin and Diversity of Life Chapter Contents 25.1

Deep Time

25.2 Origins of Life 25.3 Evidence for Early Life 25.4 Earth’s Changing System 25.5 Ever-Changing Life on Earth

Jeff Hunter/The Image Bank/Getty Images

Introduction

Visual Outline CO2 in the atmosphere CO2 combines with H2O to form carbonic acid CO2 + H2O H2CO3

Deep time

Carbonic acid reacts with rocks

Ocean

4.6 BYA

Carbon carried by rivers

Calcium and bicarbonate form calcium carbonate which precipitates Ca2+ + HCO3− CaCO3

Age of Earth >3 BYA Evidence for early life

examined in

Origin of life

Organic molecules

required

affected by Changes on Earth

Many cycles during one week

influence

Life on Earth

Heat source

Cool water

All life descended from a common ancestor and all living things have many things in common: they are composed of one or more cells, they carry out metabolism and transfer energy with ATP, and they encode hereditary information in DNA. But species are also highly diverse, ranging from bacteria and amoebas to blue whales and sequoia trees. Coral reefs, such as the one pictured here, are microcosms of diversity, comprising many life-forms and sheltering an enormous array of life. The origins and history of life on Earth are intertwined with Earth’s ever-changing geology, climate, and atmosphere. To understand the current diversity of life, we need to ask questions about the history of Earth on a geological timescale, and about how life itself affects Earth’s systems.

Eras Cenozoic

Eons

Periods Quaternary Tertiary

Mesozoic

Cretaceous

Present

50 MYA

100 MYA

150 MYA Jurassic

North and South America joined by land bridge. 0° Uplift of the Sierra Nevada. Worldwide glaciation.

Appearance of humans First primate Bird radiation Mammal radiation Diversification of flowering plants Pollinating insects

Gondwana begins 0° to break apart; interior less arid.

First flowering plants First birds

South Pole

Phanerozoic

Permian

South Pole 300 MYA

First reptiles

Paleozoic

Carboniferous 350 MYA

400 MYA

Silurian 450 MYA

500 MYA

Late Middle Early

Supercontinent of Laurentia to the 0° north and Gondwana to the south. Climate mild.

First tetrapods

Bony fish, seed plants, and insects appear Early vascular plants diversify

1000 MYA

Cambrian explosion; increase in diversity; appearance of animals First multicellular organisms

Oldest definite fossils of eukaryotes

2000 MYA

Appearance of oxygen in atmosphere

Go Gon G on o ndwa dwana dwana a Gondwana South Pole

30° 0° 30°

1500 MYA

Laurentia La Lau auren au nti tia ia ia

ana forms. Supercontinent of Gondwana rth America America. Oceans cover much of North Climate not well known. 0°

Invertebrates dominate First land plants

Ordovician

Proterozoic

a

250 MYA

Pangea intact. Interior of Pangea 0° arid. Climate very warm.

First dinosaurs First gymnosperms

Pange

Triassic

Cambrian

Gondwana Go Gon G ondwa o dwa wana na

First marsupial mammals 200 MYA

Devonian

South Pole

South Pole Gondwana Go Gon G ondwa on dw dw wana na

Rodinia South Pole

Most Mosstt of Earth E is covered in i ocean ocean and ice. ice

Cyanobacteria

Hadean

Early Middle Late

Archean

2500 MYA

3000 MYA

3500 MYA

Oldest fossils of prokaryotes

4000 MYA

Molten-hot surface of Earth becomes somewhat cooler Oldest rocks

4500 MYA

Formation of Earth

Figure 25.1 Geological timescale and the evolution of life on Earth. chapter

25 The Origin and Diversity of Life 525

25.1

Deep Time

Learning Outcomes 1. Describe the history of the Earth. 2. Understand the relationship between geological events and the evolution of life.

Earth changed over geological time Many of the fundamental questions about the history of Earth are geological. To explore the origins and diversification of life over billions of years, it is important to understand geological, or deep, time. Geological time is divided into four eons, stretching over 4.6 billion years (figure 25.1). Eons are subdivided into eras, which are further subdivided into periods. So much change has occurred since the Earth formed that no rocks exist from the first 500 to 700 million years of Earth’s history (called the Hadean eon) that preceded the earliest fossils. Although it is impossible to be certain what early Earth was like, geological evidence is consistent with a meteor hitting the Earth almost 4.6 bya with such force that that debris from the impact formed the Moon. The rocky mantle of the Earth literally melted as atmospheric temperatures exceeded 2000°C. Hadean Earth was also pummeled by asteroids, which could potentially vaporize entire oceans. Shifting between a fiery and a sometimes frozen Earth, it was a wildly dynamic environment that was unlikely to have supported life.

CO2 levels shifted, affecting temperature The early atmosphere likely had high levels of CO2, and water slowly vaporized from the molten rock. The Earth cooled over a 2-million-year period. As the temperature cooled, clouds made of silicate condensed in the atmosphere and rained down, forming a warm ocean under a CO2 atmosphere. CO2 levels dropped and the Earth cooled, and for a period of time the ocean froze. Decreases in CO2 contributed to the decrease in temperature because less radiant energy was absorbed in the atmosphere. What changed the CO2 level in the atmosphere? The ocean and atmosphere were in equilibrium in terms of CO2 levels. Volcanic eruptions added CO2 to the atmosphere and ocean, while the weathering of rock decreased CO2 levels. Weathering occurred more rapidly under hot, wet conditions than under cold, dry conditions. Weathering is the conversion of silicate rock to soil. It occurs when the CO2 in the atmosphere combines with water (H2O) to create a carbonic acid (H2CO3) rain. The carbonic acid interacted with the rock, releasing bicarbonate ions (HCO3–) and Ca2+. The weathered solutes moved through rivers and oceans and formed calcium carbonate (CaCO3), which precipitated and sequestered the CO2 in the ocean (figure 25.2).

Continents moved over geological time Earth’s crust formed rigid slabs of rock called plates under both continents and oceans. These huge slabs shift a few centimeters each year, a process called plate tectonics. The term tectonics comes from the 526 part V Diversity of Life on Earth

Greek word for “build” or “builder,” and plate movements built and continue to build the geological features of Earth. Most major earthquakes and volcanoes occur at the edges of the plates when they move. Although the plates move slowly, over deep geological time the cumulative effects are astounding. Several times in Earth’s history, all the continents have come together to form a single supercontinent (see figure 25.1). Two proposed supercontinents, Rodinia (all the continents) and Gondwana (composed of all the current Southern Hemisphere continents), existed at different times and both occupied the Southern Hemisphere. Gondwana contributed to the supercontinent Pangea, which was fully formed 225 mya and began to separate 25 to 50 million years later. Geologists have the greatest confidence in plate tectonic evidence from the last 200 million years, beginning with the breakup of Pangea.

Life emerged in the Archean At some point, life emerged. Some fossil evidence exists from the Archean eon that followed the Hadean. Two billion years into Earth’s history, the Proterozoic (meaning “early life”) eon occurred. It was characterized by the formation of the supercontinent Rodinia, which 650 mya broke up into several continents before the start of the Phanerozoic (“visible life”) eon. Collectively, the Hadean, Archean, and Proterozoic eons are referred to as the Precambrian. With the start of the Phanerozoic’s Paleozoic era, marked first by the Cambrian period, a remarkable diversification of multicellular organisms took place. Beginning with the Phanerozoic, both geologists and biologists begin to focus on chunks of time on the order of periods and shorter. The Phanerozoic eon represents only 12% of Earth’s history, yet it contains most of the biological history of the diversification of multicellular life (see figure 25.1). Birds and mammals have existed for 4% of Earth’s existence, whereas humans have existed for 0.2% of the history of Earth.

Learning Outcomes Review 25.1

The history of Earth extends over 4.6 billion years, but the geological record of what occurred during the Hadean eon before life emerged is limited. Initially, the Earth was inhospitable to life, but as conditions changed, life emerged, more than 3 billion years ago. Multicellular species appeared only within the last billion years. ■■

Given an opportunity to study the diversity of life in the fossil record, which eon would you choose? Support your choice.

25.2

Origins of Life

Learning Outcomes 1. Contrast today’s atmosphere with the likely atmosphere at the end of the Hadean eon. 2. Describe key steps necessary for life to originate.

Figure 25.2 Weathering rocks pull CO2 from the atmosphere. H2O and CO2 in the atmosphere

CO2 in the atmosphere

combine to form carbonic acid (H 2CO3), which interacts with rock to release HCO3 – and Ca 2+. These ions wash into the ocean and form calcium carbonate (CaCO3), which precipitates and sequesters the carbon in the ocean sediment.

CO2 combines with H2O to form carbonic acid CO2 + H2O H2CO3

Carbonic acid reacts with rocks

Ocean

Carbon carried by rivers

Calcium and bicarbonate form calcium carbonate which precipitates Ca2+ + HCO3− CaCO3

We really don’t know how life began on Earth. Because we cannot recreate the process now, we have to use various lines of scientific exploration to piece together the puzzle of life’s origins, beginning with the geology of early Earth. Hadean Earth was a hot mass of molten rock about 4.6 bya. As it cooled, much of the water vapor present in Earth’s atmosphere condensed into liquid water that accumulated on the surface in chemically rich oceans. One scenario for the origin of life is that it originated in this dilute, hot, smelly soup of ammonia, formaldehyde, formic acid, cyanide, methane, hydrogen sulfide, and organic hydrocarbons. Whether at the oceans’ edges, in hydrothermal deep-sea vents, or elsewhere, life likely arose spontaneously from these early waters. Although the way in which this happened remains a puzzle, we cannot escape a certain curiosity about the earliest steps that eventually led to the origin of all living things on Earth, including ourselves. How did organisms evolve from the complex molecules that swirled in the early oceans? Long before there were cells with the properties of life, organic (carbon-based) molecules formed from inorganic molecules. The formation of proteins, nucleic acids, carbohydrates, and lipids were essential, but not sufficient, for life. The evolution of cells required early organic molecules to assemble into a functional, interdependent unit.

Early organic molecules may have originated in various ways Organic molecules may have had extraterrestrial origins Organic molecules are the basis of all living organisms. How the first organic molecules formed is not known, and some could have extraterrestrial origins. Hundreds of thousands of meteorites and comets are known to have slammed into the early Earth, and recent findings suggest that at least some may have carried organic materials. For example, chemical analysis of the Tagish Lake meteorite, a rocky, carbon-based meteorite that landed in British Columbia in 2000, found that nearly 3% of the weight was organic matter. Soluble organic compounds in the meteorite included carboxylic and sulfonic acids, along with trace levels of amino acids. Glycine is the most abundant amino acid in the meteorite and its carbon isotope ratios are inconsistent with rocks found on Earth, supporting the claim that some organic molecules may have had extraterrestrial origins.

Organic molecules may have originated on early Earth There is disagreement among geochemists as to the exact com­ position of the early atmosphere. One popular view is that it was chapter

25 The Origin and Diversity of Life 527

composed primarily of carbon dioxide (CO2) and nitrogen gas (N2), along with significant amounts of water vapor (H2O). In one scenario, the early atmosphere also included hydrogen gas (H2) and compounds with hydrogen atoms bonded to other light elements (sulfur, nitrogen, and carbon) as hydrogen sulfide (H2S), ammonia (NH3), and methane (CH4). This atmospheric makeup is called a reducing atmosphere because of the availability of hydrogen atoms, with their electrons. A reducing atmosphere would not have required as much energy to drive some chemical reactions, making it easier to form the carbon-rich molecules from which life evolved. If we assume this kind of reducing atmosphere, then what kind of organic molecules would be produced by natural processes? This was first addressed by American chemists Stanley L. Miller and Harold C. Urey. In what has become a classic experiment, they attempted to reproduce the conditions in the Earth’s primitive oceans under a reducing atmosphere. Even if their hypothesis proves incorrect—the jury is still out on this—this experiment is critically important because it ushered in the field of prebiotic chemistry. Miller and Urey assembled a reducing atmosphere rich in hydrogen with no oxygen over liquid water. This was maintained at a temperature somewhat below 100°C, and provided with a natural source of energy: simulated lightning in the form of sparks (figure 25.3). They found that within a week, 15% of the carbon originally present as methane gas (CH4) had converted into other simple carbon compounds. Among these compounds were formaldehyde (CH2O) and hydrogen cyanide (HCN). These compounds then combined to form simple molecules, such as formic acid (HCOOH) and urea (NH2CONH2), and more complex molecules containing carbon– carbon bonds, including the amino acids glycine and alanine. In similar experiments performed later by other scientists, more than 30 different carbon compounds were identified, including the amino acids glycine and alanine, but also glutamic acid, valine, proline, and aspartic acid. Other biologically important

Figure 25.3 The Miller– Urey experiment. The apparatus

consisted of a closed tube connecting two chambers. The upper chamber contained a mixture of gases thought to resemble the primitive Earth’s atmosphere. Electrodes discharged sparks through this mixture, simulating lightning. Condensers then cooled the gases, causing water droplets to form, which passed into the second heated chamber, the “ocean.” Any complex molecules formed in the atmosphere chamber would be dissolved in these droplets and carried to the ocean chamber, from which samples were withdrawn for analysis.

molecules were also formed in these experiments. For example, hydrogen cyanide contributed to the production of a complex ringshaped molecule called adenine—one of the bases found in DNA and RNA. Thus, many key molecules of life could have formed in a reducing atmosphere on the early Earth.

Important landmarks in the evolution of metabolism can be identified There are two basic scenarios for the early evolution of central metabolism: an autotrophic origin, and a heterotrophic origin. In the autotrophic scenario, energy from the oxidation of inorganic compounds was used to drive a reductive version of the citric acid cycle (see chapter 7). The heterotrophic scenario builds on the MillerUrey experiment, which provides the source of a primordial soup in which life evolved. It is not possible to distinguish between these two models, and some combination of the two is also possible. It is perhaps more instructive to consider landmark events in the evolution of living systems on the Earth. These include oxygenic photosynthesis, carbon fixation, and nitrogen fixation. These metabolic events would be complemented by the evolution of information storage and expression systems involving nucleic acids and proteins. While the origin of photosynthesis is difficult to establish, the advent of oxygenic photosynthesis can be estimated based on the presence of molecular oxygen in the atmosphere. All molecular oxygen available today is the result of photosynthetic oxidation of water. As detailed in section 25.4, there were two large pulses of oxygen into the atmosphere: one in the late Archaean and one in the late Proterozoic. This implies that oxygenic photosynthesis became widespread in the late Archaean. Nitrogen fixation is important, as reduced nitrogen is critical for life: it is a limiting nutrient in most terrestrial ecosystems today. Nitrogen can be “fixed,” or made available in biologically accessible form, by the action of volcanoes, and by lightning in the atmosphere. The Miller–Urey Experiment

Electrodes discharge sparks (lightning simulation)

Water vapor

Many cycles during one week

Samples tested for analysis

Boiler

Condenser Cool water Condensed liquid with complex molecules

Heated water (“ocean”) Heat source

528 part V Diversity of Life on Earth

Reducing atmosphere mixture (H2O, N2, NH3, CO2, CO, CH4, H2)

Small organic molecules including amino acids

However, this does not produce enough fixed nitrogen to support very large populations of organisms. Biological nitrogen fixation removes this limitation. The modern enzyme required for this process is called nitrogenase, and it is found only in prokaryotes. Genomic analysis put the origin of nitrogenase at around 2.2 to 1.5 bya. More recently, an analysis of isotope ratios indicated that the origin of nitrogenase might have been earlier, in the later Archaean (2.7–2.5 bya). On the information storage and expression side, a popular model posits that an RNA world predated the modern DNA-based world. This arises from the observation that RNA can both catalyze chemical reactions (see chapter 6) and store genetic information. In this scenario RNA, rather than DNA, was the first nucleic acid that permitted self-replication, an important step toward life. Later, DNA, which is more stable than RNA, took over the information storage function. Proteins, with their greater molecular diversity, gained the enzymatic function. Although much evidence supports an early RNA world, there are some issues. One is the source of ribose on the early Earth; at present there is no good one. However, research has shown that it is possible to synthesize RNA nucleotides without pure ribose under plausible conditions on prebiotic Earth. Another challenge is how to form long chains of RNA nucleotides. One model to explain this suggests the involvement of clay surfaces, which could both concentrate nucleotides and provide a metal catalyst to link them together.

Single cells were the first life-forms In addition to metabolism, cells require membranes. Constraining organic molecules to a physical space within a lipid or protein bubble could lead to an increased concentration of specific molecules. This in turn would increase the rate of reactions involving these molecules. Although modern membranes are made up of a bilayer of phospholipids (see chapter 5), early membranes may have been composed of fatty acids. These are simpler molecules than phospholipids and were more likely to form under prebiotic conditions. Just like phospholipids, they have hydrophilic heads and hydrophobic tails so they can form bilayers and enclosed cell-like structures. At some point, these bubbles became living cells with cell membranes and all the properties of life described in the chapter introduction. For most of the history of life on Earth, these singlecelled organisms were the only life-forms. We don’t know exactly how cells formed because we can’t recreate that process, but at some point simple cellular life evolved.

Learning Outcomes Review 25.2

Whether all the organic molecules necessary for life formed on Earth or some formed elsewhere and came to Earth within meteors remains an open question. Although conditions on early Earth cannot be completely reconstructed, it is likely that the temperatures were extreme and that the atmosphere had a very different gaseous composition than it does today, which allowed organic molecules, metabolic pathways, and cells to evolve. ■■

If you could time travel back to early Earth and return with a primordial pool sample, what types of molecules would you look for to understand the origins of early life? Construct an argument for the origins of life based on your predicted molecules.

25.3 Evidence

for Early Life

Learning Outcome 1. Evaluate the strength of fossil evidence dating the origins of life.

The more we learn about the Earth’s early history, the more likely it seems that Earth’s first organisms emerged and lived at very high temperatures. By about 3.8 bya, ocean temperatures are thought to have dropped to a hot 49° to 88°C (120°–190°F). Around 3.8 bya, life first appeared, promptly after the Earth was habitable. Thus, as intolerable as early Earth’s infernal temperatures seem to us today, they gave birth to life. Here we explore the evidence for life dating back 3.2 to 3.8 billion years.

Fossil evidence indicates life originated at least 3.2 bya Early life may have arisen during the Archean, but evidence of life in the form of microfossils is difficult both to find and to interpret. Nonbiological processes can produce microfossil-like structures, and rocks older than 3 billion years are rarely unchanged by geological action over time. Two main formations of 3.5- to 3.8billion-year-old rocks have been found that are mostly intact: the Kaapvaal craton in South Africa and the Pilbara craton in western Australia. (A craton is a rock layer of undisturbed continental crust.) Structures have been found in each of these formations and others that are interpreted to be biological in origin. Although this interpretation has been controversial, the accumulation of evidence over time favors these structures as being true fossil cells. Microfossils are fossilized forms of microscopic life. Many microfossils are small (1 to 2 µm in diameter) and appear to be single-celled, lack external appendages, and have little evidence of internal structure. Thus, microfossils seem to resemble presentday prokaryotes. Currently, the oldest microfossils are 3.5 billion years old. The claim that these microfossils are the remains of living organisms is supported by isotopic data and by spectroscopic analysis that indicates they do contain complex carbon molecules. Whether these microscopic structures are true fossil cells is still controversial, and the identity of the prokaryotic groups represented by the various microfossils is still unclear. More compelling evidence comes from microfossils that are 300 µm in diameter and were part of what were microbial mats in shallow marine environments in South Africa 3.2 bya (figure 25.4). Wrinkled organic walls (160 nm thick) surrounding these car­­­­bonaceous structures can be seen with scanning electron microscopy. Transmission electron microscopy revealed hollow organic-walled vesicles between compressed walls. The features of the walls are similar to those observed in Proterozoic microfossils that are well documented to be biotic in origin. The size of 3.2-billion-year-old microfossils is consistent with eukaryotic cells, but they are more likely to be cyanobacteria (discussed shortly). chapter

25 The Origin and Diversity of Life 529

100 μm

Figure 25.4 A 3.2-billion-year-old microfossil from South Africa. Picture courtesy of E. Javaux In addition to these microfossils, indirect evidence for ancient life can be found in the form of sedimentary deposits called stromatolites (figure 25.5). These structures are commonly interpreted as a combination of sedimentary deposits and precipitated material that are held in place by mats of microorganisms. The microorganisms that make up the mats are thought to be cyanobacteria. Formations of stromatolites are as old as 2.7 billion years. Because relatively modern stromatolites are also known, the formation and biological nature of these structures is less contentious.

Isotopic data indicate that carbon fixation is an ancient process Another way to ask when life began is to look for the signature of living systems in the geological record. Living systems alter their environments, and sometimes this change can be detected. The most obvious change is that living systems are selective in the isotopes of carbon in compounds they use. Living organisms incorporate 12C into their cells before any other carbon isotope, and thus they can alter the ratios of these isotopes in the atmosphere.

They also have a higher level of 12C in their fossilized bodies than does the nonorganic rock around them. Much work has been done on dating and analyzing carbon compounds in the oldest rocks, looking for signatures of life. Analysis of carbon signatures indicates that carbon fixation, the incorporation of inorganic carbon into organic form, was active as long as 3.8 bya, consistent with the dating of the oldest microfossils. The ancient fixation of carbon happened via two main pathways. The most common pathway for carbon fixation is the Calvin cycle (see chapter 8). This is the pathway used by cyanobacteria, algae, and modern land plants that perform oxygenic photosynthesis using two photosystems. The Calvin cycle is also active in green and purple sulfur bacteria that perform anoxygenic photosynthesis using a single photosystem. This anoxygenic form of photosynthesis could account for ancient carbon fixation. To date, the entire Calvin cycle has not been demonstrated in the archaea, a group of prokaryotes, although the key enzyme for this pathway has been identified in a few archaeal isolates. Instead, some archaea use a reductive version of the citric acid cycle (see chapter 7). This pathway of carbon fixation is also used by some lithotrophic bacteria, which derive energy from the oxidation of inorganic compounds, and by the green sulfur ­ ­bacteria. Two other pathways may also occur in the lithotrophs, ­the archaea, and the green nonsulfur bacteria. Evidence suggests that the ability to fix carbon has evolved more than once over the course of evolution.

Some hydrocarbons found in ancient rocks may have biological origins Another way to look for evidence of ancient life is to look for organic molecules, which are clearly of biological origin; such molecules are called biomarkers. Although the process sounds simple, it has proved difficult to find such markers. Hydrocarbons, which are derived from the fatty acid tails of lipids, are one type of biomarker. These can be analyzed for their carbon isotope ratios to indicate biological origin. The analysis of extractable hydrocarbons from the Pilbara formation in Australia found lipids that are indicative of cyanobacteria as long ago as 2.7 billion years. The search for definitive chemical markers for living systems in the oldest rocks and in meteorites is an area of intense interest.

Learning Outcomes Review 25.3

Evidence for the earliest cells exists in microfossils. The earliest microfossils are controversial, but they are at least 3.5 billion years old. Other evidence for early life includes isotopic ratios that are skewed by biological activity. The Calvin cycle and a reductive version of the citric acid cycle, as well as other pathways, appear to have led to carbon fixation in ancient life. Some hydrocarbons appear to be biomarkers and may therefore also indicate ancient life-forms.

Figure 25.5 Stromatolites. Mats of bacterial cells that trap

mineral deposits and form the characteristic dome shapes seen here.  Horst Mahr/imagebroker/age fotostock 530 part V Diversity of Life on Earth

■■

You have discovered a fossil that may be an early bacterial cell. What evidence would be sufficient to convince you that this is indeed an early bacterial cell?

25.4 Earth’s

Changing System

0

Phanerozoic

500

Learning Outcomes

Climate (temperature and water availability) and atmosphere (including levels of CO2 and O2) are among the many factors that affect the ability of organisms to survive and reproduce. Over the course of Earth’s history, repeated and dramatic shifts in all these factors have led to mass extinctions and otherwise influenced the course of evolution (the phenomenon of mass extinction is discussed in chapter 22). Shifting tectonic plates have led to volcanic eruptions that alter the atmosphere, including simply blocking sunlight. Oscillating CO2 levels over geological time correlate with temperature changes as increased atmospheric concentrations of CO2 trap the heat radiating from the Earth, creating a greenhouse effect (see chapter 57). Some changes affecting the climate and the atmosphere are strictly geological, but living organisms account for other changes. For example, the evolution of photosynthesis increased O2 in the atmosphere. In this section, we explore how shifts in climate and the atmosphere have affected Earth and life on Earth over geological time.

Earth’s climate has been ever-changing The range of temperatures and rainfall over Earth’s history is astounding. Early Earth experienced temperatures ranging from over 2000oC at some times and global mean temperatures of –50oC at others. Temperature shifts immediately affect terrestrial, aquatic, and marine organisms. Over a slightly longer time frame, the level of oceans is affected, further disrupting life. Earth has been cooling since its formation, but several sudden drops in temperature, including three exceptionally sharp drops that occurred both early and late in the Proterozoic, have decimated life. These extreme drops in temperature resulted in glacial ice covering Earth from pole to pole, a phenomenon called Snowball Earth (figure 25.6). Under such conditions, the ice covering the Earth’s surface reflected back most of the radiant energy from the Sun, maintaining the cold temperatures. Even at the equator, temperatures would have climbed no higher than –20oC, equivalent to current Antarctic temperatures. With frozen oceans, temperatures were less moderated, and the shifts in temperature were more marked than on Earth today. Glaciation results in massive extinctions of species. It is difficult to imagine life surviving pole-to-pole glaciation, yet only 80 million years after the last Snowball Earth event, bilaterally symmetrical animals are found in the fossil record.

Geological changes and living organisms explain shifts in the atmosphere Geological changes can explain many, but not all, of the shifts in the composition of the atmosphere. Living organisms have also

Proterozoic

1500 Time (MYA)

1. Construct an explanation for the relationship between CO2 levels and glaciation. 2. Argue that plate tectonics has affected the evolution of life on Earth.

1000

2000 2500 3000

Archean

3500 4000

Hadean

4500

Solar System Origin

Reg Regional gla glaciation Sno Snowball Ear Earth

Figure 25.6 Three global glaciation events occurred during the Proterozoic. substantially altered the atmosphere, as discussed in section 25.5. Here we focus on CO2 as one example, recognizing that changes in concentration of other atmospheric gases, including O2, N2, and CH4, have affected the evolution of life on Earth. At the time of the last two Snowball Earth events in the late Proterozoic, most of the continents were in the tropics (see figure 25.1). During normal times, the hot, wet climate of the tropics accelerated weathering, described earlier, which led to a significant decline in the concentration of atmospheric CO2. The accompanying decrease in temperature slowed the weathering, stabilizing both temperature and CO2 levels. Weathering is hypothesized to have led to the late Proterozoic glaciations. In addition to changes in weathering associated with a hot, wet climate, plate tectonics can also affect weathering and thus atmospheric levels of CO2. As a large continent breaks into smaller pieces, more area is exposed to the ocean as shoreline and becomes wetter. The increased moisture leads to increased weathering and decreased atmospheric CO2 concentrations. Thus increased weathering from continental shifts during the late Proterozoic also contributed to glaciation.

Continental motion affected evolution Although they contribute to a changing atmosphere and climate, shifting plates also affect evolution by reproductively isolating populations or allowing previously separate populations to interbreed (see chapter 22). The continents sit on submerged plates that are in motion. During the Proterozoic eon, all the landmass existed as the single supercontinent Rodinia that began to break up into smaller continents about 700 mya. This and other shifts have continued throughout Earth’s history (see figure 25.1). The Paleozoic era began with the expansive diversification of life of the Cambrian period and a number of separate continents. chapter

25 The Origin and Diversity of Life 531

But, as the Paleozoic came to a close, Earth once again had a single supercontinent: Pangea. During the Carboniferous and Permian periods of the Paleozoic, major collisions of continents completed the development of the Appalachian mountains of North America, when eastern North America and northwestern Africa smashed together. Pangea lasted for 100 million years and began to break up during the Late Triassic and Early Jurassic periods of the Mesozoic era. The current Cenozoic era began 66 mya. Australia and Antarctica separated, as did Greenland and North America. The Atlantic Ocean continued to grow as plates in the mid-­Atlantic spread. Greenhouse conditions during the Cretaceous period led to a rise in sea level and extensive continental areas were submerged. During the last 2 million years, cold periods led to glaciation and a drop in sea level. As a result, a land bridge formed between Asia and North America. Humans and other animals migrated between the once disconnected continents. A connection between Australia and southeast Asia also allowed for migration. Understanding when landmasses were connected and which species existed at the time is critical in explaining the evolution of the diversity of life on Earth.

Life changes Earth Oxygenic photosynthesis produced atmospheric O2 The early atmosphere contained CO2, but, unlike the modern atmosphere, it lacked oxygen gas (O2). The evolution of photosynthesis added O2 to the ocean and to the atmosphere, providing an environment conducive to the evolution of cellular respiration (figure 25.7). Geological evidence shows a 200-million-year lag between the origins of photosynthesis and substantial concentrations of O2 in the environment. One explanation for the lag is the formation and precipitation of iron oxide in the ocean. Much of the O2 released in the ocean during the first 200 million years reacted with elemental iron to form iron oxide, which prevented an increase in atmospheric O2 concentrations. As O2 increased in the atmosphere, some of it interacted with ultraviolet (UV) radiation from the Sun and formed O3 (ozone). The ozone layer protects terrestrial Earth from UV radiation, reducing the rate of mutations and making life on land possible.

Did plants contribute to glaciations? There is growing evidence that plants contributed to two glaciations. The initial colonization of land by plants was also followed

by gradual cooling and abrupt glaciation 488 to 444 mya in the Ordovician period. The CO2 levels just prior to the cooling were in the range of 20 times higher than present-day concentrations, and climate models predict the levels would have needed to be reduced by 50% to trigger glaciation. Geological weathering could explain some cooling through decreased CO2, but the decrease would not be sufficient to trigger glaciation. The early land plants lacked roots, but studies using extant relatives show they released organic acids that can increase rock weathering. Although additional weathering caused by plants could accelerate decreases in CO2 and temperature, it would still be insufficient to trigger glaciation. The most convincing proposal is that phosphorus, an essential nutrient for plant growth, was released from the rocks through weathering and entered the ocean, where it supported extensive algal growth. The rapid growth of the photosynthetic algae led to increased uptake of CO2 for photosynthesis, decreasing atmospheric CO2 levels and triggering glaciation. Increased phosphorus in sedimentary rock from that time supports this conclusion. The entire system equilibrated after the initial release of phosphorus and plants began recycling phosphorus in the soil, eliminating the more massive runoff into aquatic environments. A second glaciation was concurrent with the diversification of vascular plants 400 to 360 mya during the Devonian period. Vascular plants’ extensive root systems increased weathering of rocks, which decreased atmospheric CO2 levels. Plant roots release the same organic acids released by the rootless early land plants that weather rocks; these essential nutrients include phosphorus. As the vascular plants colonized Earth, global cooling and glaciation at the poles followed. Plant-induced glaciation not only changed Earth, but also affected other life. Glaciation often led to rapid drops in sea level. This in turn resulted in extinction of many marine species.

Learning Outcomes Review 25.4

Changes in climate, atmosphere, and location of continents has supported the diversification of life and led to extinctions, as a result of glaciation. In concert, life has changed the Earth’s system, including land, water, and atmosphere. Both abiotic and biotic factors can lead to decreased CO2 levels, which can precipitate glaciation. ■■

Compare and contrast the events that triggered the glaciation at the end of the Proterozoic with the glaciation early in the Ordovician period.

Atmospheric oxygen level (%)

40 30

25.5

20 10

Ever-Changing Life on Earth

Learning Outcomes 3.0

2.0

1.0 Time (BYA)

Figure 25.7 Atmospheric O2 levels over time. 532 part V Diversity of Life on Earth

0

1. Compare and contrast the evolution of the endomembrane system and mitochondria. 2. Discuss the types of cell specialization necessary for multicellularity.

The progenitor for all life now on earth arose in the Archean eon. We hypothesize the existence of a last universal common ancestor, or LUCA. The descendants of LUCA include the three monophyletic clades we call domains: Eubacteria, Archaea, and Eukarya. The relationships between these three domains are still contentious, although the most common model has Eukarya branching from within Archaea. A group of deep-sea Archaean called Asgard appear to be the closest prokaryotic relatives to modern eukaryotes. The roots of the eukaryotic tree are elusive, but it does appear that the deepest branches form five so-called supergroups: Excavata, SAR (a monophyletic clade consisting of Stramenopila, Alveolata, and Rhizaria), Archaeplastida, Amoebozoa, and Opisthokonta. These groups are based on both morphological similarities and molecular analysis. They appear to form monophyletic clades, although this has not been definitively shown for all groups. The SAR group combines the older supergroup called Chromalveolata (Stramenopiles and Alveolates) with Rhizaria. These groups are shown in figure 25.8 and described in more detail later, in chapter 28. Here we will consider the key evolutionary events supporting this incredible diversity of life evolving in response to an ever-changing environment.

Compartmentalization of cells enabled the advent of eukaryotes

characterized by high temperature, high pressure, or high salt. Bacteria and archaea have some functional compartments, but eukaryotes are characterized by extensive compartmentalization. Eukaryotes appear in the fossil record only about 1.5 bya. Despite sharing basic cellular metabolism with prokaryotes, eukaryotic cells evolved structures and functions that enabled them to be larger, and eventually, allowed the evolution of multicellular life.

Evolution of the endomembrane system Eukaryotes are characterized by complex cellular organization with an extensive endomembrane system, which subdivides the cell into functional compartments, including the nucleus (figure 25.9; see also chapter 4). The innovation of a nuclear envelope segregated genetic material into its own compartment, causing the process of gene expression to be separated in time and space. Transcription in the nucleus followed by translation in the cytoplasm allows additional control over gene expression. The Golgi apparatus and endoplasmic reticulum are key innovations that facilitate intracellular transport and the localization of proteins to specific regions of the cell (see chapter 4). These membrane systems, as well as the nuclear membrane, arose through infolding of the plasma membrane. Not all cellular compartments, however, are derived from the endomembrane system.

For at least 1 billion years, bacteria and archaea ruled the Earth. No other types of organisms existed to eat them or compete with them, and their tiny cells formed the world’s oldest fossils. Archaea may be found in extreme environments, including environments Rough endoplasmic reticulum

Nucleus

Opisthokonta

Amoebozoa

Archaeplastida

Rhizaria

Alveolata

Stramenopila

Excavata

Archaea

Eubacteria

SAR

TEM 15,500×

Figure 25.8 Five supergroups have been identified within the domain Eukarya, one of three domains of life on earth. The SAR group is a monophyletic clade that joins the

older supergroups Chromalveolata and Rhizaria.

Figure 25.9 Cellular compartmentalization. These complex, single-celled organisms called paramecia are classified as protists. The yeasts, stained purple in this photograph, have been consumed and are enclosed within membrane-bounded sacs called digestive vacuoles.  Don W. Fawcett/Science Source chapter

25 The Origin and Diversity of Life 533

Molecular phylogenetic data indicate that eukaryotes arose early in the Proterozoic and diversified later in that eon. Microfossil evidence from the Proterozoic supports the existence of eukaryotes as early as 1.5 bya.

Multicellularity leads to cell specialization Brown alga

Eukaryotic cell evolutionarily incorporates red alga

Red alga

Green alga

The unicellular body plan has been tremendously successful, with unicellular prokaryotes and eukaryotes constituting about half of the biomass on Earth. But a single cell has limits, even with the within-cell specialization provided by compartmentalization in eukaryotes. The evolution of multicellularity allowed organisms to deal with their environments in novel ways through differentiation of cell types into tissues and organs. True multicellularity, in which the activities of individual cells are coordinated and the cells themselves are in contact, occurs only in eukaryotes and is one of their major characteristics. Bacteria and many single-celled eukaryotes form colonial aggregates of many cells, but the cells in the aggregates have little differentiation or integration of function (figure 25.11). Multicellularity has arisen independently in different eukaryotic supergroups. For example, multicellularity arose independently in the red, brown, and green algae. One lineage of multicellular green algae was the ancestor of the plants (see chapter 28). A different unicellular ancestor in the Opisthokonts gave rise to all multicellular animals. Multicellularity required that cells connect to each other and communicate. Although each cell has identical genetic information, gene expression varies among cells to allow specialization. Mechanisms for coordinating gene expression and cell differentiation evolved.

Cyanobacteria

Eukaryotic cell with mitochondria evolutionarily incorporates cyanobacteria

Figure 25.10 All chloroplasts are monophyletic. The same cyanobacteria were evolutionarily incorporated by multiple hosts that were ancestral to the red and green algae. Brown algae share the same ancestral chloroplast DNA, but most likely gained it by engulfing red algae.

Endosymbiosis and the origin of eukaryotes Two modern organelles, mitochondria and chloroplasts (see chapter 4), arose by the process of endosymbiosis, where a cell engulfed by endocytosis becomes part of the engulfing cell (see chapter 28). Mitochondria are thought to be derived from the parasitic bacteria Rickettsia, being incorporated into eukaryotic cells early in their history. Similarly, the chloroplasts of photosynthetic eukaryotes are derived from cyanobacteria. The red and green algae acquired their chloroplasts by directly incorporating a cyanobacterium, while brown algae most likely incorporated red algae to obtain chloroplasts (figure 25.10). 534 part V Diversity of Life on Earth

1.5 mm

Figure 25.11 Aggregation of Dictyostelium discoideum to form a colonial organism. Rupert Mutzel

tremendous diversity of life on Earth today. As the Proterozoic came to a close with the end of the third Snowball Earth event, the Paleozoic began with the Cambrian period lasting from 542 to 488 mya. This was a period of extremely rapid expansion of life, referred to as the Cambrian explosion. We know a great deal about this period because not only the hard parts of organisms, but also the soft parts were preserved in the fossil record in three sites in British Columbia, Greenland, and China (figure 25.12). For 3 billion years, life, with the exception of a few groups of  algae, had been unicellular. In the period leading up to the Cambrian radiation, the first multicellular animals appeared. During the 50  million years that followed, ancestors of almost every group of animals evolved.

Major innovations allowed for the move onto land 3 mm

Figure 25.12 Fossil from the Cambrian Explosion. An unusually large number of fossils with soft body parts, such as this Marrella splendens fossil from the Burgess Shale in Yoho National Park in British Columbia, provide evidence for the rapid diversification of animal life during this period. O. Louis Mazzatenta/

National Geographic/Getty Images

Sexual reproduction increases genetic diversity Another major characteristic of eukaryotic species as a group is sexual reproduction. Although some interchange of genetic material occurs in bacteria, it is certainly not a regular, predictable mechanism in the same sense that sex is in eukaryotes. Sexual reproduction allows greater genetic diversity through the processes of meiosis and crossing over, as you learned in chapter 13. In some of the eukaryotic supergroups, sexual reproduction occurs only occasionally. The first eukaryotes were probably haploid; diploids seem to have arisen on a number of separate occasions by the fusion of haploid cells, which then eventually divided by mitosis.

Rapid diversification occurred during the Cambrian The evolutionary innovations in cells occurred while life was primarily aquatic and established the foundations that led to the

The Cambrian radiation was confined to the ocean. Shortly afterwards, plants and then animals colonized terrestrial environments. The evolution of photosynthesis, which resulted in an O2-rich atmosphere, also resulted in the ozone layer, which protects life on the surface from UV radiation. Successfully moving from a watery to a terrestrial environment required innovations to prevent desiccation and to obtain water. Gas exchange also required new strategies. In animals, lungs were more effective than gills or gas exchange through moist skin. Plants evolved stomata, regulated openings in the surface of the plant, to facilitate gas exchange and prevent water loss. These and other major innovations are discussed later, in chapters 29 through 34.

Learning Outcomes Review 25.5

The history of life on Earth is the history of the diversification of cells and organisms. Eukaryotic cells are highly compartmentalized with endomembrane systems, and they have acquired mitochondria and chloroplasts by endosymbiosis. Multicellularity led to cellular specialization, and meiosis and sexual reproduction increased genetic diversity. Bursts of rapid radiation of species, as well as periodic extinctions, have led to the diversity of marine and terrestrial life in existence today. ■■

Analyze ways evolutionary innovations prior to the Cambrian may have contributed to the rapid radiation.

chapter

25 The Origin and Diversity of Life 535

Chapter Review Jeff Hunter/The Image Bank/Getty Images

25.1 Deep Time

Earth changed over geological time (figure 25.1). During the 4.6-billion-year history of life on Earth, Earth has experienced extreme shifts in temperature that correspond with shifting CO2 levels. Continents formed, collided to make supercontinents, and separated again multiple times. Most of the biological history of life on Earth has occurred in the last 12% of Earth’s history.

25.2 Origins of Life Early organic molecules may have originated in various ways. Some organic molecules may have had extraterrestrial origins, but most likely formed under the reducing environment of early Earth. Important landmarks in the evolution of metabolism can be identified. The origin of life may have been autotrophic or heterotrophic. Oxygenic photosynthesis evolved in the Archean, and nitrogen fixation in either the early or the late Archean. An RNA world may have preceded the modern world. Single cells were the first life-forms. At some point, simple cellular life evolved as membranes formed and bounded metabolic and self-replicating activities.

25.3 Evidence for Early Life Fossil evidence indicates life originated at least 3.2 bya. Microfossils with organic wells have been studied with scanning electron microscopy and date back at least 3.2 billion years. More substantial evidence comes from 2.7-billion-year-old stromatolites, sedimentary deposits of microorganisms. Isotopic data indicate that carbon fixation is an ancient process. Carbon dating revealed that organisms were sequestering carbon using an ancient form of photosynthesis as early as 3.8 bya. Some hydrocarbons found in ancient rocks may have biological origins. Organic molecules that clearly have biological origins, including lipids, have been identified in 2.7-billion-year-old rock formations in Australia.

25.4 Earth’s Changing System Earth’s climate has been ever-changing. Earth has been cooling since its formation, but extreme shifts in temperature, correlating with changes in CO2 levels, have been

536 part V Diversity of Life on Earth

associated with glaciation events, including three glaciations that covered the entire globe. Glaciations can lead to mass extinction and affect the course of evolution. Geological changes and living organisms explain shifts in the atmosphere. Weathering in hot, wet climates and on increased moist surface areas resulting from the breakup of supercontinents resulted in the sequestration of CO2 in the oceans. In some cases, the drop in CO2 concentrations can be sufficient to trigger glaciations. Continental motion affected evolution. Plate tectonics explains the gradual movement of continents that can change the climate and affect whether or not populations are able to mix and interbreed. Life changes Earth. Plant life has contributed to two glaciation events by pulling down CO2 from the atmosphere through both photosynthesis and the weathering of rock to release phosphorus, a necessary plant nutrient.

25.5 Ever-Changing Life on Earth Compartmentalization of cells enabled the advent of eukaryotes. Through both the infolding of membranes to form endomembrane systems and endosymbiosis, eukaryotic cells have gained compartments that enhance cellular function (figure 25.10). Multicellularity leads to cell specialization. Multicellularity arose independently in eukaryotic supergroups. Sexual reproduction increases genetic diversity. The evolution of meiosis increased the genetic diversity available for natural selection. Rapid diversification occurred during the Cambrian. The evolution of compartmentalization, multicellularity, and sexual reproduction established the foundations for the rapid evolution of animal life during the Cambrian period (542 to 488 mya). Major innovations allowed for the move onto land. Moving to a terrestrial environment required adaptations to prevent desiccation, obtain water, and facilitate gas exchange in a dry environment.

Visual Summary Deep time Age of Earth Evidence for early life

examined in

found in

Origin of life

required

Organic molecules

affected by

Fossils Changes on Earth Isotopes

arose from

influence

Hydrocarbons

modified by

Metabolism

Climate

includes

Geology

enclosed in

Temperature, water, CO2, & O2 Weathering

includes

Continental motion

includes

Life on Earth increased efficiency

changed via

Compartmentalization of cells

Sexual reproduction

led to

resulted in

Eukaryotic cells

Genetic diversity

increases atmospheric O2 Life

Single cells

Isolating or joining populations

Photosynthesis

carbon fixation

By autotrophs

nitrogen fixation

By prokaryotes

made possible Life on land

Review Questions Jeff Hunter/The Image Bank/Getty Images

U N D E R S TA N D 1. The Miller–Urey experiment demonstrated that a. b. c. d.

life originated on Earth. organic molecules could have originated in the early atmosphere. the early genetic material on the planet was DNA. the early atmosphere contained large amounts of oxygen.

2. Plate tectonics can contribute to a. b. c. d.

volcanoes and earthquakes. formation of supercontinents. increased weathering and CO2 sequestration. All of the choices are correct.

3. Identify which of the following statements is false and correct the statement. a. b.

Brown and red algae are not closely related phylogenetically. Chloroplasts in brown and red algae are monophyletic.

c. d.

Brown algae gained chloroplasts by engulfing green algae (endosymbiosis). None of the statements are false.

4. Which of the following events occurred first in eukaryotic evolution? a. b. c. d.

Endosymbiosis and mitochondria evolution Endosymbiosis and chloroplast evolution Compartmentalization and formation of the nucleus Formation of multicellular organisms

5. Fossil data indicate that a. b.

life originated 4 bya. life may have originated 3.5 bya, but definitely did so by 3.2 bya. c. the Cambrian explosion led to the origin of life. d. plants played an important role in causing glaciation.

chapter

25 The Origin and Diversity of Life 537

A P P LY 1. A global glaciation would be unlikely to occur if a. b. c. d.

a supercontinent formed near the equator and there was extensive rainfall. millions of acres of forest were cleared. vast amounts of phosphorus found its way into aquatic and oceanic environments. there was a rapid expansion of algal populations in the ocean.

2. Although we do not know how early life arose, the following likely happened: a. b. c. d.

All organic molecules were transported to Earth by meteors. High levels of oxygen were essential for glycolysis. Lipids organized to form cell membranes. Organic molecules formed once temperatures reached moderate levels, equivalent to those in today’s environment.

3. Which bits of evidence would convince you that life originated as early as 3.2 bya? a. b. c. d.

You look at a microfossil under a scanning electron microscope and it is the same shape as a cell. A high-quality transmission electron micrograph of a fossil cell reveals cellular compartments, including a possible nucleus. Potassium dating of a fossil containing a possible cell indicates that the fossil is 3.2 billion years old. Using transmission and scanning electron microscopy, you find evidence of a carbon-based material in what appears to be a cell wall of a fossil isotopically dated as 3.2 billion years old.

538 part V Diversity of Life on Earth

4. During which times would you expect that geographic isolation would be particularly important in the evolution of life? a. b. c. d.

The Cambrian period The end of the Paleozoic era The beginning of the Cenozoic era Both a and c are correct.

5. The chloroplasts of brown algae a. b. c. d.

have a chromosome with a very different DNA sequence than the chromosome of a red alga. are surrounded by four membranes. are surrounded by two membranes. have a chromosome with a DNA sequence that is similar to that in red algae, but very different from that in green algae.

SYNTHESIZE 1. Vascular plants sequestered large amounts of CO2 through photosynthesis when they first expanded across terrestrial environments, leading to a glaciation. Evaluate the claim that a similar glaciation event occurred when the first plants colonized land. Be sure to consider whether or not photosynthesis by these new species alone could cause a glaciation. 2. Synthesizing what you have learned in this chapter, analyze the multiple effects the collision of two plates could have on the evolution of life on Earth. 3. Analyze the factors that may have contributed to the Cambrian explosion.

26 CHAPTER

Viruses Chapter Contents 26.1

The Nature of Viruses

26.2 Viral Diversity 26.3 Bacteriophage: Bacterial Viruses 26.4 Viral Diseases of Humans 26.5 Prions and Viroids: Infectious Subviral Particles

Centers for Disease Control and Prevention/Cynthia Goldsmith

Introduction

Visual Outline Enveloped

36 nm

Naked

Host

classified by

Effect

can be Viruses

have DNA or RNA genome and capsid Acellular infectious agents

three types

only contain protein

Genome

Prions

are naked RNA

Viroids

Brain homogenate

PrPC null mouse

PrPC wild-type mouse Control mouse

We begin our exploration of the diversity of life with viruses. Viruses may seem like an unusual place to start as they lack many of the characteristics of life. On the other hand, their ubiquitous association with all organisms, their impact on those organisms, and their relative genetic, biochemical, and structural simplicity make them a good place to begin.     At a bare minimum, all viruses contain genetic material enclosed in a protein shell. Despite their relative simplicity, their disease-producing potential and ecological impact make viruses important biological entities. The virus particles pictured here are recreated from genomes of the influenza virus responsible for the 1918 influenza pandemic, which killed an estimated 50 to 100 million people worldwide. Other viruses cause diseases such as AIDS and the common cold, and some even cause certain types of cancer.

Viruses are obligate intracellular parasites that infect most types of cells

    Early virologists made important discoveries in molecular genetics, and their work gave us important tools used in biotechnology and genetic engineering. With the discovery of giant viruses, newly emerging viral diseases, and advances in viral ecology due to modern genomics, virology remains one of the most exciting fields in modern microbiology.

26.1 The

Nature of Viruses

Learning Outcomes 1. Describe the structures of viruses and their genomes. 2. Explain why viruses are obligate cellular parasites.

All viruses have the same basic structure: a genome of RNA or DNA surrounded by a protein shell called a capsid. If the capsid is tightly associated with the genome, then it is called a nucleocapsid. Capsids and nucleocapsids are typically composed of one to a few different protein molecules that assemble together to form predictable shapes such as icosahedrons or helical filaments (figure 26.1). Some viruses have an envelope of lipids, proteins, and glycoproteins derived from the cells they infect, and others have more complex structures. Viral particles (virions) are not cells because they generally lack metabolic activity, do not have cytoplasm, and cannot replicate outside of a host organism. Viral genomes can be linear or circular, and single- or double-stranded. Viral genomes are highly diverse and serve as one way to classify viruses, as discussed in section 26.2.

Without a suitable host, a virus cannot independently replicate its genome, or make the proteins required to assemble new virions. Also, as evolution requires transmission of genetic material, viral populations cannot undergo evolution without a suitable host. When viruses infect cells, however, they usurp normal cellular processes and acquire some of the characteristics of living things. Viruses have found suitable hosts in fungi, prokaryotes, and protists, as well as in the cells of plants and animals. Scientists have found viruses in all organisms where they have looked for them. Particular kinds of viruses can infect only particular types of cells: either the virus can recognize only one kind of cell type or it cannot replicate efficiently inside a cell type. The cell or organism types that a particular virus can infect are referred to as its host range. Within a multicellular host, many viruses also exhibit tissue tropism, targeting only a specific subset of cells. For example, Rhabdovirus, the virus that causes rabies, infects and replicates only within neurons. This tissue tropism will determine the nature of the infection and the disease symptoms caused by any particular virus.

Viruses reproduce by taking over host-cell machinery The primary function of a virus is to make more copies of itself. This function is encoded in the viral genome. Although viral genomes include genes for making capsid proteins, they often do not code for proteins involved in the expression and replication of their genomes, and never contain genes to make ribosomes. This means that viruses have to take over the host cell’s machinery to replicate their genomes, express their genes, and use the proteins produced to build new virions. In some cases, a virus must carry with it a protein needed very early in genome replication or expression. This is the case because

Antigen

RNA Capsid

DNA

RNA

DNA Capsid Tail

Structure

Capsid

Envelope

Capsid

Tropism

Plant virus (TMV)

Animal virus (adenovirus)

Bacterial virus

Animal virus (influenza)

Capsid shape

Helical

Icosahedral

Icosahedral head + helical tail

Helical (within envelope)

a.

b.

c.

d.

Figure 26.1 Structure of virions.  Viruses can be characterized as helical, icosahedral, binal, or polymorphic. a. The capsid may have helical symmetry such as the capsid of the tobacco mosaic virus (TMV). TMV infects plants and consists of 2130 identical protein molecules (green) that form a cylindrical nucleocapsid containing a single-stranded RNA genome (red). b. Icosahedral capsids, such as those of the naked adenovirus, are made from 20 triangular facets. c. Bacteriophage come in a variety of shapes, but binal symmetry is seen exclusively in phages such as the T4 phage of E. coli. This form of symmetry is characterized by an icosahedral head, which contains the viral genome, and a helical tail. d. Viruses can also have an envelope surrounding the capsid (e.g., the influenza virus). The envelope gives the virus a polymorphic shape. Within the envelope are eight RNA segments, each contained within a helical capsid. 540 part V Diversity of Life on Earth

some viral genomes are very different from their host cell’s genome. For example, HIV, which infects specific immune cells in humans, carries an enzyme called reverse transcriptase, which makes DNA from an RNA template (chapter 17). Reverse transcriptase must be carried by the virus because the single-stranded RNA genome of HIV must be converted to double-stranded, linear DNA upon infection. There is no equivalent host enzyme, so the virus must provide this enzyme for early events in infection. Once inside a cell, a virus hijacks the transcription and translation machinery to make new virus. There are three general types of viral genes expressed during infection: early genes, which contribute to taking over the host cell’s expression machinery; intermediate genes; and late genes, which are usually capsid proteins and proteins involved in viral release from the infected cell. Although an infected cell might produce thousands of new viral particles, this does not always kill the cell, and the cell can become a virus-producing factory.

Viral capsids come in two basic shapes Most viral capsids are either helical or icosahedral, although there is significant variation around these two basic structures. Helical capsids, such as that of the tobacco mosaic virus (TMV) in figure 26.1a, have a rodlike or threadlike appearance. Icosahedral capsids, such as those of adenoviruses, appear generally spherical; their icosahedral geometry is revealed only when viewed with an electron microscope (26.1b and 26.3a). The icosahedron is a structure with 20 equilateral triangular facets. Most animal virus capsids are icosahedral (figure 26.1b). The icosahedron is the most efficient symmetrical arrangement of subunits to form a shell with maximum internal capacity (figure 26.2). There is an upper limit on how large viral genomes can become if accommodated in an icosahedral capsid, and this may be the limiting factor in viral complexity. Some unusual strategies to accommodate larger genomes have been adopted by some viruses and those viruses, such as the poxviruses, have more complex structures.

Figure 26.2 Poliovirus. The poliovirus is a naked virus with

an icosahedral capsid. The capsid is formed from multiple copies of four different proteins. Image is a digitally generated 3D model. Calysta

Images/Getty Images

Viral morphology is highly diverse While capsid structure is relatively well defined, overall virus morphology is highly diverse. Viruses with no envelope, called naked viruses, will be either filamentous or icosahedral, like the poliovirus shown in figure 26.2. Some bacterial viruses, such as the T-even bacteriophage shown in figure 26.3, are complex.

Figure 26.3 A bacterial virus. 

Head DNA Capsid (protein sheath) Collar Tail

Whiskers

Base plate

a.

Bacteriophage exhibit a complex structure. a. Electron micrograph and (b) diagram of the structure of a T4 bacteriophage (some facets removed to show the DNA genome in the head). (a) Department of Microbiology, Biozentrum, University of Basel/Science Source

Tail fiber

b.

chapter

26 Viruses 541

Complex viruses have a binal, or twofold, symmetry that is neither entirely icosahedral nor entirely helical. The T-even phage in figure 26.3 has an elongated icosahedral head connected to a hollow tube with helical symmetry, ending in a complex baseplate with tail fibers. Enveloped viruses have much greater morphological diversity than naked viruses due to the flexible nature of their envelope. Many enveloped viruses, such as influenza viruses, are generally spherical but, as can be seen in the photo at the beginning of the chapter, are somewhat variable in shape. Some enveloped viruses, such as the “bullet-shaped” rabies virus, have more defined and uniform shapes due to the viral capsid that provides structure to the envelope.

The retroviruses, which include HIV, have single-stranded RNA genomes that are reverse-transcribed into double-stranded DNA by the enzyme reverse transcriptase. In HIV, the doublestranded DNA version of the genome is integrated into the host cell’s chromosomal DNA, where it is expressed to make viral proteins and new viral genomes. Other viruses, such as the viruses causing smallpox and herpes simplex, have DNA genomes. Most DNA genomes are double-stranded, and are usually replicated in the nucleus.

Viral genomes are highly diverse

Viruses are significantly smaller than cells, having diameters from about 20 to 250 nm (figure 26.4). Mimivirus, originally misidentified as a bacterium that infected amoebas, was subsequently found to be a virus 750 nm in diameter with a 1.2 Mb (megabase, or 1 million bases) genome. The identification of several additional “giant viruses” followed the discovery of Mimivirus. Pandoravirus, isolated in 2013, has micrometer-sized virions and a 2.5 Mb genome, which is larger than those of some bacteria. The largest currently known virus is Pithovirus, which was revived from 30,000-year-old Siberian ice in 2014. With virions 1.5 μm long, Pithovirus is larger than some bacteria, and even than some eukaryotic cells. Metagenomics, the study of genetic material isolated from the environment and not directly from organisms, has been used to identify a new group of giant viruses, the Klosneuviruses. The role of these giant viruses in ecosystems and disease biology is currently unknown.

Viral genomes are either DNA or RNA, linear or circular, and singleor double-stranded (table 26.1). Approximately 70% of all viruses have RNA genomes; these include the viruses that cause influenza, measles, and COVID-19. RNA genomes can be single- or doublestranded and, with one debatable exception, are linear. Because most host cells cannot make RNA from an RNA template, viral RNA genome replication uses a virally encoded RNA-dependent RNA polymerase. This polymerase is highly error-prone, which leads to high rates of genome mutation. This makes viruses with RNA genomes difficult targets for the host immune system, vaccines, and antiviral drugs. Although many RNA genomes encode genes on a single molecule of RNA, some medically important viruses, such as the influenza viruses, have segmented genomes. These genomes have several pieces of RNA, each with one to a few genes.

BACTERIUM Streptococcus 1 μm across

VIRUS Rabies 125 nm

Giant viruses challenge assumptions about viruses

VIRUS HIV 110 nm

VIRUS Influenza 100 nm

VIRUS Adenovirus 75 nm

VIRUS Poliovirus 30 nm

VIRUS Flavivirus (West Nile virus) 22 nm

VIRUS Herpes simplex 150 nm VIRUS Poxvirus 250 nm

EUKARYOTE Yeast cell 7 μm long

VIRUS T2 bacteriophage 65 nm

PROTEIN Hemoglobin 15 nm

BACTERIUM E. coli 2 μm long

Figure 26.4 Viruses vary in size and shape. Note the dramatic differences in the sizes of cells and viruses, and the diversity of viral morphologies.

542 part V Diversity of Life on Earth

TA B LE 2 6 .1

Important Human Viral Diseases

Disease

Pathogen

Genome

Vector/Epidemiology

Chicken pox

Varicella-zoster virus

Double-stranded DNA

Spread through contact with infected individuals. No cure. Rarely fatal. Vaccine available since 1995. Latent infections may cause shingles; a higher dose of the ‘95 vaccine is available to protect against shingles.

Hepatitis B (viral)

Hepadnavirus

Double-stranded DNA

Highly infectious through contact with infected body fluids. Approximately 1% of U.S. population infected. Vaccine available. No cure. Can be fatal.

Herpes

Herpes simplex virus

Double-stranded DNA

Can cause epithelial blistering; spread primarily through skin-to-skin contact with cold sores/blisters. Very prevalent worldwide. No cure. Exhibits latency— the disease can be dormant for several years.

Mononucleosis

Epstein–Barr virus

Double-stranded DNA

Can cause extreme fatigue and flulike symptoms that can persist for several weeks but possibly longer. Spread via bodily fluids, especially saliva. Infection may result in an increased likelihood of some rare cancers.

Cervical and penile cancer

Human papillomaviruses 16/18

Double-stranded DNA

HPV is the most common sexually transmitted infection in the United States. HPV types 16 and 18 can cause cervical and penile cancers. Other subtypes can cause genital warts. A safe and effective vaccine is available for HPV 16/18.

AIDS

HIV

(+) Single-stranded RNA (two copies)

Destroys immune defenses, resulting in death by opportunistic infection or cancer. In 2013, WHO estimated that 35 million people are living with AIDS, with an estimated 2.1 million new HIV infections and an estimated 1.5 million deaths.

Zika virus disease

Zika virus

(+) Single-stranded RNA

Transmitted by the bite of infected Aedes mosquitoes. Can be transmitted mother to fetus during pregnancy. Symptoms of infection, if any, include fever, rash, malaise, headache. Complications during pregnancy can cause microcephaly and other congenital abnormalities. No vaccine exists.

West Nile fever

Flavivirus

(+) Single-stranded RNA

Spread by mosquitoes and can be amplified in bird hosts. Can lead to neurological problems. Present in U.S. since 1999.

Ebola

Filoviruses

(−) Single-stranded RNA

Acute hemorrhagic fever; virus attacks connective tissue, leading to massive hemorrhaging and death. Peak mortality is 50–90% if untreated. Outbreaks confined to local regions of central Africa.

Influenza

Influenza viruses

(−) Single-stranded RNA (eight segments)

Historically a major killer (20–50 million died during 18 months in 1918–1919); wild Asian ducks, chickens, and pigs are major reservoirs. Vaccines, which have to be remade each year, are available.

Measles

Paramyxoviruses

(−) Single-stranded RNA

Extremely contagious through contact with infected individuals. Vaccine available. Usually contracted in childhood, when it is not serious; more dangerous to adults.

SARS

SARS-Cov

(+) Single-stranded RNA

Identified in early 2003; spread via aerosolic droplets of saliva. Causes severe acute respiratory syndrome with roughly 3% mortality.

COVID-19

SARS-Cov-2

Rabies

Rhabdovirus

First reported in late December 2019, based on a cluster of cases of disease in Wuhan province, China. Commonly causes fever, dry cough, fatigue, but other symptoms including loss of taste or smell, conjunctivitis, and skin rashes. Caused a global pandemic starting in 2020. (−) Single-stranded RNA

An acute viral encephalomyelitis transmitted by the bite of an infected animal. Fatal if untreated. Commonly infected animals include bats, foxes, skunks, and raccoons. Domestic animals can be infected.

If size alone is not enough to challenge assumptions about the distinction between viruses and cells, the genomes of some of these giant viruses also contain features not previously observed in viral genomes. For example, before the discovery of the giant viruses, no viral genome encoded proteins involved in translation. The genomes of the isolated giant viruses collectively possess nine of the 20 aminoacyl-t-RNA synthetase enzymes, and the hypothetical Klosneuvirus genome contains genes for all 20. The presence of parts of the translation machinery in the giant viruses suggested the hypothesis that they represented a fourth Domain in the Tree of Life, sharing a cellular ancestor with the other domains. However, phylogenetic analysis of the gene sequences of the aminoacyl-tRNA synthetase genes revealed that the giant viruses were not descended from a cellular ancestor. Rather, the evidence suggests that they acquired translation-related genes from hosts as once smaller viruses.

Learning Outcomes Review 26.1

Viral capsids have relatively simple structures, being either icosahedral, helical, or a combination of both. Virion morphology is much more diverse, however. Viruses reproduce by taking over the machinery of an infected cell, using its proteins for genome replication and expression. Viral genomes can be DNA or RNA, although most are RNA, and can be single- or double-stranded. Some RNA genomes are segmented. Giant viruses may be as large as or larger than some bacteria, and have genomes similarly sized. ■■

The capsid of naked viruses is assembled from many copies of only a few different proteins. How might this keep the genomes of these viruses very small?

26.2 Viral

Diversity

Learning Outcomes 1. Compare different classification systems for viruses. 2. Describe the role played by metagenomics in describing viral diversity.

Despite the fact that all viruses are obligate intracellular parasites, viruses show significant diversity. This diversity is the basis for several different virus classification schemes. Viruses are most commonly classified based on taxonomy, the type of disease the virus causes, the host range or tissue tropism of the virus, or genome type and expression. Different kinds of classification systems meet the needs of different kinds of scientists: a medical microbiologist might be most interested in a classification based on disease type, while a molecular geneticist may find more utility in a classification based on genome expression. The use of metagenomics to identify novel genomes, like that of Kloseneuvirus, makes it increasingly important that viruses are classified and can be referenced consistently. The choice of a classification system should consider whether it 544 part V Diversity of Life on Earth

allows predictions about group members given the characteristics of the group.

Taxonomic classification of viruses A taxonomic classification of viruses has been produced by the International Committee on Taxonomy of Viruses (ICTV). This taxonomy uses the conventional systematic groups of order, family, subfamily, and genus, but omits the groups of kingdom, class, and phylum. DNA viruses can often be assigned to species groups; however, this is more problematic for the RNA viruses. Numerous criteria, such as host range, virion morphology, and genome type, are considered when placing a virus in the taxonomy. When genes are conserved between different viruses, phylogenetic analysis can also guide where a virus is placed in a taxonomy.

Classification systems based on disease or host It is attractive to classify viruses based on disease because of familiarity with viral diseases like the flu or ebola. However, such a classification suffers from a major weakness: by definition it excludes any virus that doesn’t, or doesn’t appear to, cause disease. Moreover, some viruses cause different diseases in different contexts, and some viruses cause disease in some individuals and not in others. For example, the varicella-zoster virus that often causes chickenpox in children causes a different disease— shingles—in adults that have previously had chickenpox. In the years between having chickenpox and having shingles, a person harbors the virus but displays no disease symptoms. Classifying varicella-zoster virus by the disease it causes could require that it be classified in two different places in a taxonomy, defeating one of the purposes of classification. Other diseases, the common cold for example, can be caused by many different viruses, including rhinoviruses, adenoviruses, and coronaviruses. Placing these viruses together in a group because they cause the same disease may be suggestive that they have other characteristics in common—such as genome architecture—when they don’t. Host-based classifications group viruses in terms of their host range and are as deceptively attractive as disease-based classifications. Some viruses infect very different types of organisms. Consider the difficulty of classifying influenza viruses based on host. There are four types of influenza virus (A, B, C, D), and the different types can cause varying degrees of symptoms, or no symptoms at all, in animals as diverse as primates, pigs, poultry, bats, cats, dogs, and cows.

Classification based on genome expression A less intuitive, but possibly more powerful, approach to viral classification considers how viral genomes are expressed. The Baltimore classification, developed by Nobel laureate David Baltimore, overcomes many of the problems of disease- or host-based classifications. The classification uses the way viral genomes are replicated and expressed to create seven groups of viruses, with each known virus being placed into only one group (figure 26.5).

Group I Group II Group III Group IV Group V Group VI Group VII DNA (+/–) DNA (+) RNA (+/–) RNA (+) RNA (–) RNA (+) DNA (+/–) Reverse transcription

DNA (+/–)

RNA (–)

RNA (+) Reverse transcription

viral genome introduced into a cell cannot serve the demand for making all that protein. By making thousands of minus-sense RNA copies of a plus-sense RNA genome, thousands of plussense RNA molecules can then be made and used to direct protein synthesis of new viral proteins for virion assembly; those plus-sense RNA molecules can also be used as new genomes for virus production. As mentioned briefly earlier, viruses with RNA genomes that do not have a DNA intermediate in their replicative cycles must use a special enzyme to replicate and express their genomes. Viruses with RNA genomes express RNA-dependent RNA polymerases so they can convert an RNA genome, of plus- or minus-sense orientation, into an RNA strand of the opposite polarity. This allows the virus to either make mRNA for translation or make templates for the production of new viral genomes. Group VI viruses are the retroviruses. These viruses have plus-sense single-stranded RNA genomes, which have to be converted into double-stranded DNA to be expressed and used to make new RNA genomes. Group VII contains only a handful of viruses, which have double-stranded DNA genomes, with gaps in some of the DNA strands, so parts of the genome are single-stranded.

Genomics is driving advances in viral ecology mRNA

Figure 26.5 The Baltimore classification of viruses.

Classification is on the basis of the relationship between genome organization and production of mRNA for gene expression. (+) and (−) indicate the sense of the strands of nucleic acid. Plus-sense strands have the same sequence as mRNA, and minus-sense strands are the complement of plus-sense strands.

Group I viruses have double-stranded DNA that use host machinery to replicate and express their genomes. Group II viruses have single-stranded DNA genomes and must convert them to double-stranded DNA to be expressed and replicated in host cells. Group III viruses have double-stranded RNA genomes. One strand of the genome is transcribed to produce mRNA that can be translated by host cell machinery. Because many host cells cannot create RNA using information in an RNA template, the enzyme to do this must often be carried inside the virus. Group IV and Group V viruses have plus (+) sense  and minus (–) sense single-stranded RNA genomes respectively. Like mRNA, plus-sense RNA can be translated to make protein. Minus-sense RNA is the complement of plus-sense RNA and cannot be translated. A minus-sense RNA genome must be converted to plus-sense RNA by a viral protein before it can be used for translation, or to make new copies of the genome. Even when a virus has a plus-sense genome, it must be used to make many minus-sense RNA copies. This is because to make thousands of new virions during an infection, the viral genome has to direct the host cell to make large amounts of viral proteins. The single

Only a small fraction of all viruses cause disease in animals or plants. The majority of viruses infect prokaryotes and certain protists (called protozoans), which play critical roles in energy and matter transformations in ecosystems. Given that the majority of prokaryotes and protozoa cannot be cultivated in the lab, studying their viruses is virtually impossible. In turn, this means that we cannot study the majority of viruses and the impacts they have on their hosts and ecosystems. Metagenomics has provided a partial solution to this problem. Metagenomics involves sampling soil or water (or even air) from an environment and then isolating DNA from the sample. The isolated DNA is then amplified using PCR and sequenced (see chapters 17 and 18). The sequences found in the environmental sample can be compared to characterized sequences in DNA databases to identify the species present in that environment. When this approach yields sequences not found in the databases, a novel species might have been found, even without isolating the actual organism. Viral metagenomics has identified thousands of new kinds of viruses, and ICTV has taken the unusual step of allowing the classification of species identified only by metagenomics. Metagenomic estimates indicate that approximately 1031 viral particles exist at any given time in the biosphere. In one liter of coastal seawater, there are 10 times more viruses than bacteria, which makes viruses possibly the largest source of genetic diversity on the planet. These viruses are predicted to be establishing 1024 infections per second, and every infection has the potential for horizontal gene transfer, a significant mechanism in driving rapid evolutionary change. It is impossible to anticipate the impact that human behaviors will have on viral populations, and thus on ecosystems biology, until we know more about viral ecology. chapter

26 Viruses 545

Learning Outcomes Review 26.2

Viral diversity makes it challenging to choose classification criteria. Taxonomic classifications group similar viruses using several criteria and establish a uniform nomenclature. Diseaseand host-based classifications are useful in limited situations, while criteria using how genomes are expressed and replicated are more broadly applicable. Metagenomics is expanding our understanding of viral diversity and the roles of viruses in ecosystems. ■■

The genome of influenza viruses is composed of eight pieces of minus-sense RNA. Into which of the Baltimore classification groups would influenza viruses be placed?

26.3 Bacteriophage:

Viruses

Bacterial

Learning Outcomes 1. Distinguish between lytic and lysogenic cycles in bacteriophage. 2. Describe how viruses can contribute DNA to their host.

a characteristic “head-tail” morphology that is found only in bacterial viruses (see figure 26.3). Although they are rarer, there are also bacteriophage with only icosahedral or helical capsids, which can be enveloped or naked. E. coli phage were among the first discovered and remain among the best-studied viruses. One category of these viruses is called the “T” series (T1, T2, etc.). To illustrate the diversity of these viruses, T3 and T7 phage are icosahedral and have short tails. In contrast, the so-called T-even phages are more complex, with an icosahedral head, a neck with a collar and “whiskers,” a long tail, and a complex base plate (see figure 26.3).

Bacterial viruses exhibit two replication strategies Viral infection usually results in the production of new virus particles, which when released may kill the host cell. Released virions can infect other cells, perpetuating an infectious cycle. This kind of infectious cycle is called a lytic cycle because the virus usually causes the cells to rupture, or lyse. Some bacterial viruses, called temperate viruses, can also enter a lysogenic replicative state, which usually involves the incorporation of their genome into the host cell’s genome. These genomes do not produce virions, do not kill the host cell, and are transmitted from cell to cell when the cell divides. Certain triggers can cause a lysogenic infection to become a lytic one, with the resulting death of the host cell.

The lytic cycle Bacteriophage, or phage for short, are structurally and functionally diverse viruses that infect only bacteria. Most bacteriophage have

Figure 26.6 Comparison of lytic and lysogenic cycles. 

Lytic Cycle Attachment: virus attaching to cell wall

The lytic cycle of virus reproduction is illustrated in figure 26.6. The basic steps of a phage lytic cycle are similar to those of a

Bacterial chromosome

Penetration: viral DNA injected into cell

In the lytic cycle the viral DNA directs the production of new viral particles by the host cell until the virus kills the cell by lysis. In the lysogenic cycle, the bacteriophage DNA is integrated into the host chromosome. This prophage is replicated along with the host DNA as the bacterium divides. It may persist as a prophage, or enter the lytic cycle and kill the cell. Bacteriophage are not drawn to scale in this diagram. Lysogenic Cycle Integration: of genome leads to prophage

Release: lysis of cell

Propagation: of prophage along with host genome

Synthesis: protein and nucleic acid

Cell stress

Assembly: involves spontaneous assembly of capsid and enzyme to insert DNA

546 part V Diversity of Life on Earth

Reproduction of lysogenic bacteria

Induction: prophage exits the bacterial chromosome, viral genes are expressed

naked animal virus. Although the basic outline of this infective cycle is common to most viruses, the details vary, as do the viruses themselves. The first step is attachment (or adsorption): The virus contacts and becomes specifically bound to the cell. This step limits both the host range and the tissue tropism of viruses, as viral surface proteins will only bind to specific proteins on the surface of host cells. The second step is penetration: the virus introduces its genome into the host cell through the bacterial cell wall. This has been studied in detail in phage with head–tail morphology, such as bacteriophage T4. Once contact is established, the tail sheath contracts, and the tail tube passes through an opening that appears in the base plate, piercing the bacterial outer membrane and then the cell wall. The viral genome is passed through the contracted tail into the cytoplasm of the host cell. Once inside the host cell, the viral genome takes over the cell’s replication and protein synthesis machinery in order to synthesize viral structural components. These are then assembled (assembly phase) to produce mature virus particles. In the release phase, mature virus particles are released, either through the action of virally encoded enzymes that lyse the host cell or by budding through the host cell wall.

The lysogenic cycle The most common form of lysogeny occurs when a temperate phage infects a host cell, and rather than entering a lytic cycle, its genome is integrated into the genome of the host cell. This integration allows a virus to be replicated along with the host cell’s DNA. The integrated phage genome is called a prophage, and the cell containing a prophage is called a lysogen. In a lysogen, expression of the prophage genome is repressed (see chapter 16). Although the lysogen is quite ­stable, it can be induced to enter the lytic cycle (figure 26.6, right). If the cell is stressed, the prophage can be derepressed, expressing the e­ nzymes necessary for excision of the genome, and the lytic cycle can ­commence. The switch from lysogeny to a lytic cycle is called induction because it requires turning on—inducing—the genes necessary for the lytic cycle (see chapter 16).

Lysogeny can change host phenotype During lysogeny, viral genes may be expressed from the prophage at the same time as host cell genes. When the phenotype or characteristics of the lysogenic bacterium are altered by gene expression from the prophage, the change is called phage conversion. One example of phage conversion occurs during all lysogenic infections: the host cell is resistant to subsequent infection by phage of the same type. Because many infections are lytic, this affords an obvious selective advantage on the host cell. Due to the number of bacterial species that can be lysogenized, lysogeny likely plays an important role in bacterial evolution. Sometimes, phage conversion can change an otherwise benign bacteria into a highly pathogenic strain.

Phage conversion of the cholera-causing bacterium Vibrio cholerae is usually a relatively benign, curved bacterium found living in water. However, several disease-causing strains

are also known. If ingested, these strains cause the disease cholera. Each year cholera causes acute diarrhea in about 3 million people, killing approximately 120,000. Cholera is transmitted mainly via fecally contaminated drinking water and is, therefore, most dangerous to the 2 billion people who lack access to clean water. Disease-causing strains of the cholera bacterium are created when a benign strain becomes lysogenized by the CTXϕ virus. The genome of CTXϕ encodes two proteins that together make a toxin called CTX (cholera toxin). The toxin genes are expressed along with host genes when CTXϕ lysogenizes a host; this converts a harmless bacterium into a deadly pathogen. The discovery that CTXϕ initiates an infection by attaching to the surface of Vibrio by binding to hair-like projections called pili (see chapter 27) has influenced vaccine development. The pili are also required for the attachment of the bacterium to the epithelia of the small intestine, where the severe diarrhea is caused. The expression of the toxin genes and the pilus genes always occurs together, so that pathogenic Vibrio both express the toxin and make pili at the same time. Current vaccines using killed strains of Vibrio that do not express either pilus genes or toxin genes are ineffective, provide only shortlived protection, or have adverse side effects. Research suggests that vaccines based on killed strains of Vibrio that express pilus genes would be more effective than other vaccines.

Learning Outcomes Review 26.3

Phage have two possible replicative cycles: a lytic cycle that results in cell lysis, and a lysogenic cycle where the viral genome is integrated into the host genome, creating a lysogen carrying a prophage. The prophage is transmitted vertically by cell division. The lytic cycle can be induced in a lysogen by environmental conditions. A prophage can contribute genes to the host. The toxin produced by V. cholerae and responsible for the disease cholera is encoded by a prophage. ■■

What might be the benefit of lysogeny to lysogenic phage?

26.4

Viral Diseases of Humans

Learning Outcomes 1. Describe the differences between acute and persistent infections. 2. Relate antigenic drift and antigenic shift to seasonal flu outbreaks and pandemics. 3. Exemplify chronic and latent viral infections.

chapter

26 Viruses 547

The flu is caused by influenza viruses

To establish an infection and cause disease, a virus must first have access to tissues in the body. Although skin is exposed, and therefore seems to be most vulnerable to infection, the most common point of infection is respiratory epithelia. Skin is a protective barrier to infection because it has multiple layers—the outermost of which is keratinized—and is slightly acidic. Viruses can breach the skin barrier where it is broken by injury, via feeding insects, and by damage caused during medical procedures. In addition to lacking the protective features of skin, respiratory epithelia have a surface area about 70 times that of the skin, and are exposed to a continuous flow of potentially virus-containing air as you breathe. Viruses can also gain entry to the body at the gastrointestinal mucosa, the surface of the eye, and the urogenital epithelia. Once a virus has infected a cell at the site of entry, it must replicate in that cell and may cause disease, either there or after it has spread to secondary sites. There are two main mechanisms by which a virus can spread: in blood or lymph, or via neurological tissue. In blood and lymph, viruses may be associated with immunological cells or be freely circulating, a condition known as viremia. Viral infections causing disease can be categorized as acute or persistent based on how rapidly and frequently virus is produced and on the appearance of associated symptoms. Persistent infections can be latent or chronic; however, there is variation in these classifications (figure 26.7).

Influenza in humans is caused mainly by either the influenza A or the influenza B virus, with influenza A being more clinically important. These are the viruses you are vaccinated against if you get the seasonal flu vaccine. Influenza viruses are enveloped, type V viruses spread via direct transmission of sputum in coughs and sneezes, through inhalation of aerosolized virions, or by contact with a virus-contaminated surface. Influenza virions attach to a specific surface carbohydrate on respiratory epithelial cells, using a viral envelope protein called hemagglutinin (HA). The virus is then endocytosed into the host cell’s cytoplasm. The eight segments of the viral genome are released from the capsid and transported into the nucleus. The viral genome is replicated and used to make mRNA from which viral proteins are translated. Viral capsids containing the genomic segments are assembled at the cell membrane and a virion buds from the cell surface. The viral envelope is created from the host cell membrane. Release of the virus from the cell surface depends on a viral protein embedded in the host cell membrane called neuraminidase (NA) (figure 26.8a). Disease is partly due to tissue damage caused by the virus itself, but is mainly due to the inflammatory response of the host. HA and NA in the viral envelope are the two viral proteins most commonly recognized by the immune system. As inflammatory cells of the immune system respond to the virus, infected cells are killed, and this contributes to tissue damage. While extremely unpleasant, most cases resolve without intervention; however, in patients with weakened immune systems, disease can be severe, leading to respiratory failure and death. Ironically, a very strong immunological response can be as dangerous as a weak one. The strength of immune responses triggered by the 1918 influenza A virus may explain the high mortality rate during that pandemic.

Acute viral infections lead to rapid symptom onset Acute infections involve the rapid replication of virus, which can lead to the sudden onset of symptoms. Either these infections are controlled by the immune system and resolve, or they cause the death of the host. Although there are exceptions, acute infections are often established at the site of infection and are non-life-threatening. Given the amount of virus produced, the site of production, and typical symptoms, viruses causing acute infections are easily spread in the air and are commonly responsible for outbreaks, epidemics, and pandemics.

Figure 26.7 Progression of acute and persistent infections. An infectious agent can create a.

Replication of the single-stranded, segmented RNA influenza genome uses a viral RNA-dependent RNA polymerase. This enzyme a.

b.

Infection

an acute infection, where disease onset is rapid and correlates with the peak of viral load, or may cause b. a persistent/chronic type of infection, where after initial mild symptoms, the agent persists at low levels until more serious disease occurs later as viral load increases. Some viruses can cause c. latent infections, where after an acute disease episode due to high viral load, the virus will remain dormant in the host, emerging periodically to cause symptoms. Prions and cancer-causing viruses can be categorized as d. slowly progressing agents which do not cause disease until significantly after the initial infection.

Antigenic drift reduces vaccine effectiveness

c.

Many years

Acute e.g. Influenza viruses, Rhinovirus.

Disease

Chronic e.g. HIV, Hepatitis viruses.

Many years Disease Disease

d.

548 part V Diversity of Life on Earth

Disease

Disease

Latent e.g. Herpesviruses.

Slowly progressing agents e.g. Prions, Cancer-causing viruses.

and NA proteins. If you are infected with a strain similar to the one you are vaccinated against, your immune system will quickly recognize and control the virus. If you are unfortunate enough to become infected against an influenza virus that has different variants of those proteins, you will not be protected, and the disease will run its course as your immune system mounts a slower response.

Mutation

Replication (new viruses produced) Host has immunity to influenza virus strain

Mutations during viral replication cause mutations that allow virus to evade immunity

a.

Avian influenza virus

Antigenic shift can cause flu pandemics

HA spikes NA spikes

NA RNA HA RNA

Human influenza virus

NA RNA

HA RNA

Co-infection of pig by avian and human viruses HA

NA

Novel influenza virus with avian influenza NA spikes and human influenza HA spikes

b.

Figure 26.8 Genetic drift and genetic shift. a. Genetic drift leads to small changes in HA or NA structure such that antibodies from an adaptive immune response to a prior infection cannot recognize a new strain of influenza virus. b. Genetic shift occurs only in influenza A viruses. Co-infection of a host by two influenza A strains can lead to genomic reassortment and the production of new strains with new host range.

makes approximately one mutational error per replicated genome. The high mutation rate leads to antigenic drift in the HA and NA genes. Antigenic drift refers to small changes in the HA and NA proteins such that previous vaccine-induced immunity is no longer protective (figure 26.8a). Because antigenic drift is due to random mutations, it is impossible to predict precisely which viral strains will emerge each year. Immunologists and epidemiologists at the Centers for Disease Control and Prevention (CDC) use information on circulating strains and epidemiological data to make the best possible predictions for which strains to use in next year’s flu vaccines. When you receive a flu vaccination, you are protected against certain strains of influenza virus. The vaccine primes your immune system to recognize influenza viruses with certain HA

While antigenic drift causes minor changes in the HA and NA proteins, a process called antigenic shift can cause major changes. Antigenic shift happens only in influenza A viruses, because only they have the ability to infect a wide range of animal hosts. Antigenic shift occurs when an individual is infected with more than one virus, and the viral genomes are reassorted during infection. Antigenic shift producing new HA–NA combinations was responsible for the three major flu pandemics of the 20th century. The “Spanish flu” of 1918, A(H1N1), killed 50 to 100 million people worldwide. The Asian flu of 1957, A(H2N2), killed over 100,000 Americans. The Hong Kong flu of 1968, A(H3N2), infected 50 million people in the United States, with 70,000 deaths. The first pandemic of the 21st century started in February of 2009, and by mid-May 11,000 people in 41 countries had been affected. Modern genomics provides us a unique view into how the virus causing this pandemic arose. In the 1990s, three influenza viruses infected pigs: a classical swine flu virus, a North American avian influenza virus, and a human H3N2 virus. When all three viruses infect the same cell, the eight genomic segments from each virus are all replicated. When new viruses are assembled, the eight genomic segments are selected randomly, which can shuffle the genomes from different viruses to produce new combinations of genome segments. Because the HA and NA genes are on different genome segments, this can produce viruses with new combinations of HA and NA proteins. Such a recombined virus circulated in swine herds in North America, but could not be transmitted to humans. Sometime later, pigs harboring the new virus were infected by an avian-like influenza virus. This allowed a second round of reassortment in the pigs, to create a new virus. This virus, called H1N1/09, or more commonly “swine flu,” could now infect humans and cause disease. This reassortment created a virus with an HA protein much better suited to binding to human respiratory epithelia (figure 26.8b).

Three coronaviruses cause moderate to severe disease in humans Coronaviruses are enveloped viruses with plus-sense singlestranded RNA genomes. Three coronaviruses cause moderate to severe disease in humans: MERS-CoV, SARS-CoV, and SARSCoV-2. Coronaviruses are zoonotic: they can infect a variety of nonhuman animals and occasionally they spill over from their nonhuman hosts into humans, where they can spread rapidly to cause epidemics or pandemics. There are seven coronaviruses that infect humans, four of which cause relatively mild coldlike symptoms. Of the other three coronaviruses that cause disease in humans, SARS-CoV and SARS-CoV-2 are the most important. SARS-CoV first appeared in southern China in late 2002 and spread to create an epidemic in about 24 countries before being contained in 2003. No cases of SARS have been reported since chapter

26 Viruses 549

2004. SARS-CoV causes severe respiratory disease characterized by fever, general flulike symptoms, and possibly a dry cough. Many of those infected develop pneumonia. The virus is spread by close contact, most likely via respiratory droplets expelled during coughing or sneezing, but can also be spread when objects contaminated by aerosolic droplets from an infected person are touched by the uninfected. SARS-CoV-2 was identified as the causative agent of coronavirus disease 2019 (COVID-19) in the Wuhan province of China in January 2020. The virus spread globally, causing about 1 million infections in the three months from January to March 2020. By June 2020, the number of infections globally reached 10 million, causing about 500,000 deaths. By mid-November 2020, the World Health Organization was reporting over 58 million infections and nearly 1.4 million deaths globally. The Centers for Disease Control and Prevention was reporting 12 million cases of infection and over 250,000 deaths in the United States alone. Towards the end of 2020, pharmaceutical companies in the United States, Europe, and Russia began to report vaccines providing 70–95% protection against infection. SARS-CoV-2 infects respiratory epithelia by binding, via its surface spike proteins, to the ACE2 protein found on the surface of cells in the respiratory tract (figure 26.9). Once inside the cell, the viral genome is replicated and expressed, and new virions are assembled. New, infectious virions are released from the cell by exocytosis and can infect nearby cells, spreading the infection. Individuals infected with SARS-CoV-2 exhibit a range of morbidities. Many are asymptomatic, but others develop systemic symptoms such as chills, fatigue, fever, myalgia, and malaise. Others exhibit a range of severities of respiratory tract manifestations: sinusitis, rhinitis, cough, and chest pain. In severe cases the disease progresses to cause acute respiratory distress, pneumonia, kidney failure, and death. Some individuals develop neurological symptoms such as dizziness and headache, and in some cases, there are digestive complaints. In individuals that clear the infection and recover in 2–3 weeks, there can be long-term complications from infection: heart palpitations, carditis, impaired lung function, hair loss and skin rashes, and certain psychiatric manifestations such as depression and anxiety. Understanding COVID-19 is made even more difficult because of a highly variable infection-to-fatality ratio (IFR, the relationship between how many people get infected and how many die from a disease) as a function of age: the IFR is lowest for children aged 5–9 years old and increases linearly from about 0.05 for children 10–14 years old to over 1.0 for adults 65 and older. There is also significant variation in the IFR when considering other demographic variables (e.g., by country and by biological sex).

Persistent viral infections can be chronic, latent, or slow In persistent viral infections, the virus or its genome can be detected in the host for long periods of time, usually years; in many cases, the persistence is lifelong. Based on the amount of virus present and when it is produced, these infections can be classified as chronic, latent, or slow. These classifications are complicated by how and when symptoms manifest. We will use Human Immunodeficiency Virus (HIV), the virus causing AIDS, and herpesvirus infections to illustrate chronic and latent infections. 550 part V Diversity of Life on Earth

HIV infection is chronic HIV transmission occurs when contaminated bodily fluids come into contact with a mucous membrane, or directly enter tissues. The two most common routes of HIV transmission are intimate sexual contact and the sharing of needles by intravenous drug users. Like many chronic infections, the initial stages of HIV infection follow acute infection dynamics. During the acute phase, a high viral load is characterized by as many as 5 × 106 virions per mL of blood plasma. At this stage, virus is spreading throughout the body, establishing infections in many tissues; the adaptive immune system is not yet responding to the infection and antibody-based tests for infection appear negative. Symptoms of this stage, often called “acute retroviral syndrome,” are typically flulike but may include diarrhea and joint pain. About three months after infection, the adaptive immune system begins to control the infection and the number of viruses in the blood drops dramatically. The immune system may control the replication of the virus for many years. The low viral load during this time is characteristic of a chronic infection, and those infected are generally asymptomatic. Ultimately, however, the immune system cannot control the virus and as viral load increases, the symptoms of AIDS appear (see figure 26.7b).

HIV infection compromises the immune system HIV infects and ultimately destroys certain types of immune cells. Immune cells are characterized based on cell-surface proteins. The cells targeted by HIV are cells that express the antigen CD4, and are called CD4+ cells. The most important cell types infected by HIV are T-helper cells, which regulate the immune response (chapter 50), and macrophages, which engulf nonself materials such as pathogenic bacteria. HIV infects and kills the CD4+ cells until very few are left. Without these crucial immune system cells, the body cannot mount a defense against invading pathogens. AIDS patients die of infections that are usually benign. These diseases, called opportunistic infections, are part of the progression from being HIV infected to having AIDS. The CDC defines AIDS based on the level of CD4+ cells in blood. A person is said to have AIDS when the CD4+ T-cell count drops below 200/microliter of blood or 14% of the total T-cell population, and when the person has at least one of the HIV-associated diseases or syndromes.

HIV replication involves reverse transcription HIV is a retrovirus: it has two identical, single-stranded, plus-sense RNA genomes that must be converted to double-stranded DNA by reverse transcription before being expressed and replicated. Reverse transcription, you will recall, is the use of an RNA template to make a piece of complementary DNA. Reverse transcription uses the enzyme reverse transcriptase, which is not encoded by the human genome and so must be carried into cells by the virus itself (see chapter 17). In the case of HIV, the DNA version of the genome is inserted into the host cell genome. Without this step, viral replication cannot occur. Figure 26.10 summarizes the infectious cycle of HIV. Embedded in the envelope of HIV, there is a glycoprotein called gp120 that recognizes and binds to CD4 on target cells. Successful infection also requires either the CCR5 or the CXCR4 coreceptor on the host cell surface. Once CD4 and a coreceptor are engaged by gp120, the viral envelope and the cell membrane are drawn close

10 Exocytosis

1

SARS-CoV-2

Binding of virus spike protein to ACE2 receptor on cell surface

ACE2

2 Endocytosis

3

9

Budding of immature virion from Golgi/ERGIC membrane and maturation

Release of viral genome from capsid and endosome into cytosol

Ribosome (+) sense RNA genome

ERGIC 4

5

Translation to produce viral RNA-dependent RNA polymerase (RdRp)

Replication of viral genome by RdRp

Viral genome

(+) sense RNA genome for virions

Translation of nucleocapsid proteins at endoplasmic reticulum

(+) sense transcripts for viral structural proteins (-) sense copy of genome

Transcription of (-) sense genome to make (+) sense genomes for virions and transcription of genes for viral structural proteins

Assembly of (+) sense RNA genome, and nucleocapsid protein at surface of Golgi/ERGIC

Nucleocapsid Spike Endoplasmic reticulum (ER)

Membrane 6

8

7a

Envelope

7b

Co-translation of spike, membrane, and envelope proteins at endoplasmic reticulum

Figure 26.9 SARS-CoV-2 infection and replication mechanism. (1) Virions attach to ACE2 receptors on the host cell surface;

(2) receptor-mediated endocytosis brings the virion into the cell; (3) virions are released from endosomes and the viral (+) sense RNA genome is released from the viral capsid into the cytoplasm of the cell; (4) host cell ribosomes translate the viral genome to make an RNA-dependent RNA polymerase (RdRp), which (5) makes (−) sense copies of the viral genome; (6) the (−) sense copies of the viral genome can then be copied into thousands of (+) sense RNA molecules, some of which encode the (7a) N protein which is translated in the cytosol and (7b) the S, M, and E proteins that are inserted into the ER membrane by co-translation; (8) viral genomes are assembled with the N protein to form capsids at the membrane of either the Golgi or an ER-derived compartment called the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) which contains the viral envelope proteins; (9) immature virions bud from the surface of the Golgi or ERGIC and mature inside vesicles before (10) being exocytosed.

together and fuse. The two nucleocapsids with their enclosed genomes are spilled into the cell. Once inside the cell, the genomes are released from their nucleocapsids and converted to double-stranded DNA by reverse transcriptase. Reverse transcription is inaccurate and produces at least one new mutation per viral genome replicated. Some estimates suggest that there is more genetic diversity in the viruses of an HIV-infected individual than in all of the influenza viruses worldwide! This variation creates serious problems in the treatment of HIV infection, and in the development of HIV vaccines, as we discuss below.

The double-stranded genomes are carried into the nucleus of the host cell with a viral enzyme that integrates them randomly into the host chromosomes. Transcription and translation of the viral genomes produces envelope, capsid, and several other proteins involved in viral assembly. New RNA genomes are produced by transcription of the integrated viral DNA, and are assembled into immature virions at the inner leaflet of the cell membrane. Interactions between the cell membrane and some of the viral proteins cause budding of the virion. The viral envelope, with its embedded proteins, is chapter

26 Viruses 551

Envelope

HIV

gp120 glycoprotein

and ends with free HIV particles present in the bloodstream of the human host. These free viruses infect white blood cells that have CD4 receptors, cells called CD4+ cells.

2. The viral contents enter the cell by endocytosis.

Ruptured capsid

1. The gp120 glycoprotein on the surface of HIV attaches to CD4 and one of two coreceptors on the surface of a CD4+ cell.

CCR5 or CXCR4 coreceptor

Figure 26.10 The HIV infection cycle. The cycle begins

Entry into CD4+ Cells

Attachment

CD4+ cell

Viral RNA Replication an nd Assembly and

4. The double-stranded DNA is then incorporated into the host cell's DNA by a viral enzyme.

Nucleus Transcription RNA

CD4 receptor

5. Transcription of the DNA results in the production of RNA. This RNA can serve as the genome for new viruses or can be translated to produce viral proteins.

Reverse transcriptase

Viral RNA

Host cell's DNA

DNA Doublestranded sstrand str st trand anded anded ed DNA NA A Ribosome R boso Rib osome o some e

3. Reverse transcriptase catalyzes, first, the synthesis of a DNA copy of the viral RNA, and, second, the synthesis of a second DNA strand complementary to the first one. Virus exits by budding.

RNA genomes Assembly 6. Complete HIV particles are assembled and HIV buds out of the cell. As the disease progresses, HIV-infected T-helper cells, but not macrophages, are killed by a poorly understood mechanism.

derived from the host cell membrane as the virus buds from the cell surface. HIV, like other enveloped viruses, does not lyse infected cells. However, when viral loads and replication rates are high, the stresses on infected T cells and macrophages can result in their death. This, in combination with virally induced programmed cell death, is responsible for the reduction in immune function that leads to the establishment of opportunistic infections that often lead to death in AIDS patients.

HIV treatment targets several aspects of the infection Early regimes for treating HIV infection used only a single drug. Due to the high mutation rate during reverse transcription, resistance to some of these drugs appeared quickly, and spread rapidly throughout HIV populations. The advent of highly active antiretroviral therapy (HAART), which used multiple drugs at once, largely overcame the problem of resistance to single drugs. In many cases, if the timing of treatment is appropriate, the depletion of CD4+ cells late in infection can be effectively reversed using HAART and disease progression can be slowed or even halted. Anti-retroviral drugs target different aspects of the viral replicative cycle (figure 26.11). Early drugs focused on blocking the process 552 part V Diversity of Life on Earth

of reverse transcription. Other drugs interfere with viral attachment, the integration of the viral genome into the host cell genome, and the viral assembly, maturation, and budding processes. Resistance to an anti-retroviral drug arises because the drug represents strong selective pressure on the virus. Mutations in the viral genome that render the drug ineffective are selected for, and these mutations will spread in the viral population. One drug, Maraviroc, has the unusual property that it does not target the virus. Maraviroc binds to the CCR5 receptor on the host cell and blocks attachment of the viral envelope protein gp120 so that viral attachment and entry is blocked. Resistance to Maraviroc is highly unlikely for two reasons. First, because rates of mutation in the host cell are orders of magnitude lower than in the virus, the chances of a CCR5 mutation that blocks Maraviroc binding are incredibly low. Second, there is no selective advantage to a cell with a CCR5 mutation that interferes with Maraviroc binding; subsequently, there is no pressure selecting for that mutation in the host cell. There are two well-documented reports of people living with HIV (PLWH) having been cured of their HIV infections following bone marrow transplants. For example, Timothy Ray Brown was diagnosed as being HIV-positive in 1995. In 2007, Mr. Brown developed acute myeloid leukemia. In an attempt to treat the cancer,

Coreceptor Blocking Attachment Coreceptor antagonists

Transcription

Host cell DNA

Polypeptide

Viral RNA

Integration

Integrase

Virus-cell fusion Blocking Entry Fusion inhibitors

Translation

Reverse transcriptase inhibitors (NRTI, NNRTI)

Blocking Maturation Protease inhibitors

Integrase inhibitors

Reverse transcriptase Blocking Replication

Blocking Integration

Processing by viral protease

DNA DN D NA A CD4+ cell

Figure 26.11 Viral targets for therapeutic drugs. A simplified version of the HIV infection cycle is shown, with the steps targeted for drug intervention. Five parts of the infectious process have been targeted: viral attachment, viral entry, viral genome replication, integration of the viral genome into the host genome, and viral maturation. NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor. Mr. Brown received a bone marrow transplant from a donor who had a rare mutation in the CCR5 coreceptor that makes cells resistant to infection by HIV. Mr. Brown was later declared to be cured of HIV. Despite the bone marrow transplant and his cure of HIV infection, Mr. Brown tragically died from a relapse of leukemia on September 29, 2020, aged 54. Sixty-three other PLWH have been found to have been cured of the infection; however, this is due to the virus being contained in a part of the host cell’s genome that is not expressed.

Latent viral infections are lifelong infections Viruses establishing persistent infections have the advantage that they can exist in the host population for long periods of time. In contrast, viruses causing acute infections may be eliminated from a population before new susceptible hosts become available. Persistent viral infections that are latent are characterized by an initial acute phase of infection followed by periods of no or very low viral load, and intermittent episodes of viral replication and disease.

Herpes virus infections There are eight herpes viruses, all of which establish latent infections in humans. Herpes simplex viruses 1 and 2 (HSV-1; HSV-2) are endemic worldwide and over 4 billion people are infected. Upon initial infection with HSV-1 or HSV-2, there is an acute episode of disease at the site of infection, characterized by the formation of painful blisters. Upon infection, both viruses replicate rapidly in cells at the site of infection. Infection evokes an innate immune response, which in itself causes some tissue damage. After this initial

response, an adaptive immune response is triggered that resolves the infection. During the time the immune response takes to resolve the disease, virus can migrate from the site of infection to the endings of sensory nerves innervating the infected tissue. The virus travels along the axon of an infected neuron to the body of the cell, where the virus establishes a latent infection in the nucleus of the neuron. HSV-1, which primarily causes orofacial herpes, will migrate to the trigeminal ganglia, a collection of neurons located just in front of the ear. HSV-2, which is primarily responsible for genital herpes, will move to the sacral ganglia located at the base of the spine in the lower back (figure 26.12). HSV-1 and HSV-2 can remain hidden in their respective ganglia because the viruses do not produce many proteins and infected neurons do not trigger strong immune responses. The mechanism of viral latency remains unclear; however, it is largely controlled by a single viral mRNA and several microRNAs (miRNAs) that suppress viral genome expression. There is little advantage to a virus in remaining perpetually latent. HSV-1 and HSV-2 can leave their latent state in the neurons of ganglia in response to certain environmental triggers. Suspected triggers of latent virus reactivation include changes in hormones in the body, exposure to UV light, and immune suppression. When a herpes virus is reactivated, new viruses are produced, which travel back down neurons to the original site of infection, where new lytic infections are established (figure 26.12). This leads to an outbreak of disease characterized by the same (but milder) symptoms that were seen in the initial acute infection. Given the prevalence of intimate contact between humans, this is most likely when the virus will spread to a new host. chapter

26 Viruses 553

Latency established in neurons of ganglia

Reactivation

Sacral ganglia

Human Papilloma Virus (HPV) and cervical cancer

Sacral ganglion

Urogenital epithelia

Lesions caused by HSV

Figure 26.12 Establishment of latency by herpes simplex virus. Initial infection produces an acute episode of disease, involving blister formation and a robust immunological response. Virus can enter neurons and travel via retrograde transport from the dendrite to the nerve body housed in ganglia. Poorly understood physiological triggers can cause virus reactivation. Virus travels via anterograde transport to the originally infected epithelia, where disease is triggered by viral replication.

Cancer-causing viruses are rare but often deadly Several viruses are known to contribute to cancer in humans. Some of these viruses cause cancer only in severely immunocompromised individuals, such as AIDS patients. Virally induced cancers can be caused in a variety of tissues, which largely reflect the site of infection, or a site to which the virus can disseminate following infection.

CDK/cyclin

pRB E2F

Genomic stress

Although there is great diversity in the types of virus causing cancer, most have certain characteristics in common. First, virtually all use DNA as their genetic material at some time in their replicative cycle. Second, all have some mechanism for making sure that their genome is stably inherited during cell division. Third, all must effectively evade the immune system to establish some kind of persistent infection, given the length of time that cancers take to develop. HPV is the most common sexually transmitted infection and certain “high-risk” types of HPV cause a variety of cancers in humans. Possibly the most significant of these is cervical cancer, of which approximately 70% of cases are caused by HPV infection. While some of the HPVs that infect the anogenital area are benign and cause only wartlike growths on the skin, infections of mucosal epithelia by HPV 16 and HPV 18 can cause normal cells to behave like cancer cells. This transition is slow and usually associated with the integration of the viral genome into the host cell genome. Expression of all but two of the viral genes is suppressed: The E6 gene and the E7 gene are necessary to drive tumorigenesis. E6 and E7 are viral proteins that interfere with the normal regulation of the cell cycle and apoptosis. During a normal cell cycle (see chapter 10), the retinoblastoma protein (pRb) controls the ­activity of a transcription factor called E2F. When pRb is bound to E2F, progress through the cell cycle is prevented. When cellular conditions are right for cell growth and division, pRb releases E2F, which then promotes cell cycle progression (figure 26.13a, upper panel). The viral E7 protein binds to Rb, releasing E2F regardless of whether cell division should occur. This forces cell division when an infected cell is receiving signals to not divide (figure 26.13b, upper panel). Fortunately, a fail-safe mechanism exists to prevent this kind of aberrant cell division: if levels of E2F become abnormally high, as they do when E7 forces release of E2F from Rb, a host cell protein called p53 triggers apoptosis (figure 26.13a, lower panel). However, the virus can overcome this “fail-safe” mechanism: the viral E6 protein can bind to and inactivate p53 and prevent it from triggering cell

Cell cycle progression

E7

Cell cycle progression

E6

p53 DNA damage

a.

Apoptosis

pRB

Cell cycle progression

E2F

p53 degradation p53

p p53

Cell cycle progression

Apoptosis

b.

Figure 26.13 E6 and E7 proteins of HPV drive tumorigenesis. a, upper panel. Retinoblastoma (pRb) normally binds E2F to prevent

cell division. In response to growth signals, pRb releases E2F to promote cell division. b, upper panel. HPV E7 protein binds to pRb, disrupting E2F binding. Released E2F stimulates cell division. a, lower panel. DNA damage and other genomic stress leads to accumulation of p53, which stops cell division and triggers apoptosis. b, lower panel. HPV E6 protein binds to p53 and stimulates p53 degradation. In the absence of p53, cells cannot trigger apoptosis or stop cell division. Source: Tod Duncan, University of Colorado Denver.

554 part V Diversity of Life on Earth

death (figure 26.13b, lower panel). Uncontrolled cell division in the absence of apoptosis to regulate cell number is often an early step in tumorigenesis. There are effective vaccines for some of the cancercausing types of HPV, which can significantly reduce the risk of cancer due to HPV infection.

Emerging and re-emerging viral diseases are an increasing public health risk An emerging disease is one that appears in a population for the first time, or whose geographic spread or incidence is increasing. A re-emerging disease is one that caused significant illness or death in the past, and whose incidence is on the increase after a period of low incidence. Diseases can emerge due to interactions between social, economic, and biological variables. For example, changes in sexual behavior or drug use may facilitate the spread of a virus, or an increase in global travel due to prosperity may allow viruses access to new susceptible hosts. Alternatively, genetic change, such as that seen each year in certain influenza viruses, can create strains not easily defeated by the immune system. As humans expand into environments where animals carrying viruses harmful to humans live, there is an increased risk of transmission of viruses between nonhuman animals and humans. In some cases a virus may jump a species barrier that it previously respected. For example, HIV emerged into human populations when a related virus, simian immunodeficiency virus (SIV), infected humans from a monkey host (see section 23.5). Unfortunately, emerging and re-emerging diseases often take healthcare professionals, governments, and epidemiologists by surprise. An outbreak of hemorrhagic fever caused by the Ebola virus in 2014–2015 spread rapidly through West Africa, becoming pandemic before a response could be mounted to control the disease. The Ebola virus has a mortality rate of 25 to 70% and with approximately 20,000 infections globally, resulted in more than 10,000 deaths.

Learning Outcomes Review 26.4

Viral infections are acute or persistent. In humans, influenza viruses cause acute disease. Their genomes change due to antigenic drift and antigenic shift, causing seasonal outbreaks and pandemics, respectively. Several coronaviruses can cause moderate to severe disease in humans: SARS-CoV caused a limited epidemic in 2002–2003; SARS-CoV-2, which causes the disease COVID-19, caused a pandemic that started in 2020, which had killed 1.4 million people worldwide by mid-November 2020. HIV is a chronic infection that leads to destruction of the immune system, creating susceptibility to life-threatening opportunistic infections. Latent viruses, such as HSV-1 and HSV-2, often cause acute disease and then become dormant, causing disease only when intermittently reactivated. HPV 16 and HPV 18 can cause cervical cancer by causing uncontrolled cell proliferation and suppressing apoptosis. ■■

Why can influenza viruses, but not HIV, undergo antigenic shift?

26.5 Prions

and Viroids: Infectious Subviral Particles

Learning Outcome 1. Explain how prion replication and transmission violates traditional notions of heredity.

Transmissible spongiform encephalopathies (TSEs) are an unusual group of diseases characterized by the presence of numerous small cavities in the brains of affected individuals. These cavities are caused by the loss of neurons, and can take decades to form following the initial infection by the causative agent. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) or “mad cow” disease, chronic wasting disease in deer, and kuru and Creutzfeld-Jakob disease (CJD) in humans. TSEs can be transmitted experimentally by injecting infected brain tissue into a recipient animal’s brain. TSEs can also spread via tissue transplants and, apparently, by consumption of tainted food. In the late 20th century, the disease kuru was discovered among members of the Fore people of Papua New Guinea. Members of the Fore group, primarily women and children, engaged in ritualistic consumption of their dead. This led to the spread of a commonly fatal TSE through the tribes. Because women engaged in the practice more than men, the disease resulted in a significant gender imbalance in the tribes. Mad cow disease spread widely among the cattle herds of England in the 1990s because cows were fed bone meal prepared from sheep and cattle carcasses to increase the protein content of their diet. Like the Fore, the British cattle were eating the tissues of animals that had died of the disease. In the years following the outbreak of BSE, there has been a significant increase in CJD incidence in England. Some cases appeared to be genetic; however, some patients with no family history of CJD were being diagnosed with the disease. This led to the discovery of a new form of CJD called variant CJD (vCJD) that is acquired from eating the meat of BSE-infected animals. Concern grew that vCJD may be transmitted from person to person through blood products, similar to the transmission of HIV through blood and blood products.

TSEs are caused by prions: proteinaceous infectious particles In the 1960s, British researchers Tikvah Alper and John Stanley Griffith noted that infectious TSE preparations remained infectious even after exposure to radiation that would destroy DNA

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26 Viruses 555

or RNA. Griffith then suggested that the infectious agent was a protein. This suggestion was not accepted by the scientific community, because it violated a key tenet of molecular biology: only DNA (or RNA in some viruses) acts as hereditary material. In the early 1970s, American physician Stanley Prusiner began to study TSEs. Like Griffith, he concluded that the infectious agent was a protein that he named a prion, for “proteinaceous infectious particle.” Prusiner was eventually able to isolate a distinctive prion protein that he convincingly showed was responsible for TSEs. Every host tested to date expresses a normal prion protein (PrPC) in its cells. Disease is caused by a misfolded version of PrPC called PrPSc. In vitro the misfolded PrPSc acts as a scaffold to drive PrPC to misfold into PrPSc. PrPSc is very resistant to degradation, making it possible for it to pass through the acidic digestive tract intact and be transmitted by ingesting PrPSc-­ contaminated food. Experimental evidence has accumulated to support this model of prion disease. Injection of prions with different ­abnormal conformations into hosts leads to the same abnormal conformations as in the parent prions. Mice genetically engineered to lack PrPC do not develop disease if infected with brain tissue from a mouse harboring PrPSc (figure 26.14). If brain tissue with the prion protein is grafted into the mice, the grafted tissue—but not the rest of the brain—can then be infected with TSE. PrPC is a GPI-anchored protein that is translated at the endoplasmic reticulum (ER) and transported to the surface of neurons through the endomembrane system, where it performs a poorly characterized function. If PrPC interacts with PrPSc at the cell surface, PrPC is rapidly converted to PrPSc, which leads to aggregation of the protein into fibrils and plaques. These protein aggregates interfere with cell function, trigger cell death, and cause neurodegeneration that leads to the characteristic symptoms of the diseases.

SCIENTIFIC THINKING Hypothesis: PrPC is required for scrapie pathogenesis. Prediction: PrPC null mice infected with PrPSc should not develop scrapie. Test: Construct PrPC null mice. Inject them, and PrPC wild-type mice, with brain homogenates from PrPSc-infected mice.

PrPSc-infected mouse

Brain homogenate

PrPC wild-type mouse Control mouse

PrPC null mouse

Mice live normal life span. Brain appears normal.

Mice die of neurological disorder. Brain sections show characteristic damage.

Result: All control mice died with scrapie-like neurological damage. Periodic sacrifice and histological examination showed normal disease progress. None of the PrPC null mice showed any sign of neurological disorder. Conclusion: Normal PrP protein is necessary for productive infection by scrapie infectious particle. Further Experiments: What kind of experiment would conclusively prove that the infectious agent of scrapie is a misfolded PrPC protein?

Viroids are infectious naked RNA molecules Viroids are naked molecules of circular RNA, ranging in size from about 250 to 400 nucleotides, some of which cause severe disease in economically important crops. Viroids infect plants via breaks in the epidermis, or by being injected into cells by feeding insects such as aphids. Viroid replication uses a plant cell RNA polymerase that is able to make RNA molecules from an RNA template. The polymerase makes single RNA molecules that contain repeats of the viroid genome that self-cleave into individual viroid genomes using an RNA catalytic activity specific to the RNA molecule. Mature viroids travel from cell to cell in plants via plasmodesmata. During viroid replication, small interfering RNAs (siRNAs) may be produced; these can interfere with plant growth and development to cause mild to severe disease.

556 part V Diversity of Life on Earth

Figure 26.14 Demonstration that PrPSc is required for scrapie pathogenesis.

Learning Outcomes Review 26.5

Prions and viroids are smaller and simpler than viruses. Prions are infectious particles that do not contain any nucleic acid. The pathogenic form of a prion appears to be a misfolded version of a normal cellular protein. Prions cause a number of TSEs in animals. Viroids are infectious RNA molecules that can cause severe disease in some plants. ■■

Given that pathogenic prions can be passed from one organism to another to cause disease, what kind of information do they carry that results in the change in phenotype?

Chapter Review Centers for Disease Control and Prevention/Cynthia Goldsmith

26.1 The Nature of Viruses Viruses are obligate intracellular parasites that infect most types of cells. Viruses have either an RNA or a DNA genome that is surrounded by a capsid made of protein. Viruses can be enveloped or naked. Viruses require a host to proliferate. Once inside a host, they take over the host cell machinery to reproduce. Viruses have a specific host range, and exhibit tissue tropism. Viral replication involves viral gene expression, viral assembly, and virion release from the host cell. Some viruses contain special enzymes required to initiate replication. Viral capsids come in two basic shapes. Viral capsids are generally either icosahedral or helical, but variations exist (figures 26.1, 26.2, and 26.3). Virions are structurally diverse, with enveloped viruses being more diverse than naked viruses. Some bacterial viruses are complex, with helical and icosahedral structures. Viral genomes are highly diverse. Viral genomes may be DNA or RNA, linear or circular, and single- or double-stranded (table 26.1). RNA viruses may have segmented or nonsegmented genomes. Retroviruses contain RNA that is transcribed into DNA by reverse transcriptase. Giant viruses challenge assumptions about viruses. Mimivirus and Pandoravirus exemplify the giant viruses, which are as large as small bacteria. These viruses and other giant viruses contain elements of the translational machinery, which were likely acquired during viral evolution.

26.2 Viral Diversity Taxonomic classification of viruses. The ICVT uses several criteria to create a viral taxonomy that is updated regularly, and which includes viruses identified by metagenomics. Classification systems based on disease or host. Viruses can be classified based on the disease they cause or the host they infect. These systems have limited utility, as not all viruses cause disease or they may cause different diseases under different conditions or at different times during infection. Other diseases may be caused by many different viruses. Classifications based on host range are also problematic: some viruses infect different kinds of organisms. Classification based on genome expression. The Baltimore classification sorts viruses based on the relationship between genome structure and genome expression (figure 26.5). Genomics is driving advances in viral ecology. Metagenomics can identify viral genomes without the need to isolate the viruses in the lab. Advances in metagenomics have revealed important roles for viruses in ecology and evolution.

26.3 Bacteriophage: Bacterial Viruses Bacterial viruses exhibit two replication strategies. The lytic cycle kills the host cell, whereas the lysogenic cycle incorporates the virus into the host genome as a prophage (figure 26.6). A cell containing a prophage is called a lysogen. The prophage can be induced by DNA damage and other environmental cues to reenter the lytic cycle.

For most phage, steps in infection include attachment, injection of DNA (penetration), macromolecular synthesis, assembly of new phage, and release of progeny phage. Lysogeny can change host phenotype. Phage conversion happens when gene expression from a lysogenic virus changes host cell phenotype. In addition to driving bacterial evolution, it can sometimes convert a harmless bacterium into a deadly pathogen.

26.4 Viral Diseases of Humans Acute viral infections lead to rapid symptom onset. Viruses that replicate rapidly at the site of infection can cause rapid onset of disease and are often spread in the air (figure 26.7a). Influenza is caused by influenza A and B viruses. Both are RNA viruses with segmented genomes. Antigenic drift causes minor changes in HA and NA proteins and produces seasonal flu (figure 26.8a). Antigenic shift produces strains of virus with novel combinations of HA and NA, and can cause pandemics (figure 26.8b). Three coronaviruses cause moderate to severe disease in humans. SARS-CoV and SARS-CoV-2 have caused the most severe morbidity and mortality of the coronaviruses that infect humans. SARS-CoV caused an epidemic in 2002–2003. SARS-CoV-2, which causes COVID-19, had infected 58 million and killed 1.4 million individuals by mid-November 2020. Both viruses can cause fatal upper respiratory tract infections, and SARS-CoV-2 can cause other long-term complications. Persistent viral infections can be chronic, latent, or slow. Some viruses produce chronic, latent, or slowly progressing infections with symptoms often appearing many years after the initial infection (figure 26.7b, c). HIV infection is chronic. Following an initial increase in viral load and mild symptoms, HIV is controlled by the immune system, persisting at low levels in immune tissues. As HIV slowly destroys immune cells, viral load increases and the host becomes susceptible to opportunistic infections. HIV is a retrovirus that uses reverse transcription to convert its RNA genome into DNA that become integrated into the host cell genome (figure 26.10). Treatment of HIV infection involves cocktails of drugs that target a variety of steps in the infection cycle (figure 26.11). Latent viral infections are lifelong infections. Viruses that “hide” in the host to evade the immune system can establish lifelong latent infections. HSV-1 and HSV-2 trigger acute disease episodes and then become latent inside neurons. Physiological stimuli can trigger their reactivation and lead to additional disease episodes (figure 26.12). Cancer-causing viruses are rare but often deadly. Some viruses can cause cancer. HPV initiates tumorigenesis by blocking cell death and by promoting cell division (figure 26.13). Emerging and re-emerging viral diseases are an increasing public health risk. Viruses may cause emerging or re-emerging disease due to socioeconomic changes, changes in urbanization and population dynamics, and evolution of viral genomes. chapter

26 Viruses 557

26.5 Prions and Viroids: Infectious Subviral Particles

reaction of misfolding in normal proteins that leads to the formation of protein aggregates that cause disease (figure 26.14).

TSEs are caused by prions: proteinaceous infectious particles. TSE disease-causing prions (PrPSc) are the same protein as the normal version (PrPC), but misfolded. The misfolded form catalyzes a chain

Viroids are infectious naked RNA molecules. Viroids are circular, naked molecules of RNA that infect plants. They use host protein to replicate.

Visual Summary Enveloped

consisting of

Lipids proteins glycoproteins

Naked

Host

classified by

Effect

bacteriophage infect

Bacteria

can be Viruses

Acute infection Humans

have DNA or RNA genome and capsid Acellular infectious agents

reproduce using

only contain protein

three types

are naked RNA

Genome

Prions

Host machinery

misfolded proteins

replication strategies

infect

Viroids

Latent infection Chronic infection

informs

PrPc→PrPsc Plants

to cause

Viral ecology

Lysogenic cycle

Lytic cycle

Review Questions Centers for Disease Control and Prevention/Cynthia Goldsmith

U N D E R S TA N D 1. Why do retroviruses need to contain the enzyme reverse transcriptase? a. b. c. d.

Because host cells make DNA from RNA only in G2 of the cell cycle, and HIV infects only during G1 of the cell cycle. Because host cells’ genomes do not contain a gene encoding an enzyme that can make DNA from RNA. Because HIV deletes large parts of the host cell genome upon infection, and that means there is a chance that host cell reverse transcriptase will be deleted. Host cells cannot make RNA from DNA.

558 part V Diversity of Life on Earth

2. What is the most reasonable explanation for why a bacteriophage capable of infecting E. coli would be unable to infect a human lung epithelial cell? a. b. c. d.

The nature of the genetic material is different in lung cells from that in E. coli. Bacteriophage need the reverse transcriptase enzyme that is found only in E. coli. The receptors to which the bacteriophage attach on E. coli are not found on lung cells. The bacteriophage is too large to enter the bronchioles of the lungs.

3. Why might the ability of a virus to convert a benign bacterium into a pathogen be selected for by natural selection? a. b.

The pathogenic virus will kill the host. The pathogenic virus may cause disease symptoms (diarrhea, coughing) that lead to spreading of the virus. c. The pathogenic virus may not respond to antibiotics. d. The pathogenic virus will be more stable in the environment. 4. Which of the following kinds of proteins would be least likely to be expressed during the lysogenic cycle of a temperate phage? a. b. c. d.

A protein to integrate the prophage into the host genome A protein to switch off genes for the lytic cycle of the phage A protein to suppress DNA replication in the host A protein to promote cell lysis by breaking down the cell wall of the host cell

5. Why is a drug that blocks HIV binding to one of its cell surface receptors not effective at treating influenza? a. b. c. d.

HIV and influenza viruses have different receptors to which they bind. HIV and influenza viruses use different types of genetic material. The mechanism for genome replication in HIV and in influenza viruses is different. HIV causes a chronic infection; influenza causes an acute infection.

6. Why is HAART more effective than a single drug in reducing HIV levels in infected people? a. b. c. d.

The drugs together are more toxic to the virus than is any one drug alone. Mutations that confer resistance to several drugs are much less likely than is a mutation that confers resistance to a single drug. The drugs work synergistically to boost the immune system. They work together to reduce antigenic shift in the viral genome.

7. The “information” contained in PrPSc must be contained in a. b. c. d.

the sequence of nucleotides in the PrPC gene. the sequence of nucleotides in the PrPSc gene. The sequence of amino acids in the PrPSc polypeptide. The three-dimensional structure of the PrPSc protein.

A P P LY 1. In very rare cases, HIV can infect T cells that revert to what is called a memory state. In this state, HIV genes are not expressed. At a later time, the T cell may become permissive for HIV gene expression and new virions are made. What kind of viral infection does this behavior of HIV exemplify? a. b. c. d.

Acute viral infection Slowly progressing viral infection Chronic viral infection Latent viral infection

2. What viral strain could be created by the coinfection of an H2N2 human influenza A virus and an avian H3N8 human influenza A virus into a pig? a. b. c. d.

H8N2 H3N2 H2N3 None of the above

3. What characteristic of the enzyme used by influenza viruses to replicate their genomes is responsible for genetic drift and is also a

characteristic shared by the enzyme used in the replication of the HIV genome? a. b. c. d.

Both work on an RNA template. Both produce DNA. Both make mistakes at high rates. Both are carried inside the virion.

4. How does the use of one or only a few proteins to make a viral capsid help to keep viral genomes small? a. b. c. d.

Only one or a few genes are needed in the genome. Capsid genes are small. Capsid proteins can be assembled into complex shapes. Exons do not need to be removed from the capsid genes.

5. Which approach would be best to identify novel viruses in a wastewater treatment plant? a. b. c. d.

Collecting random samples of wastewater and trying to culture individual viruses by infecting them into a range of different cell types Metagenomics on samples of wastewater 16S rDNA PCR amplification and DNA sequencing 18S rDNA PCR amplification and DNA sequencing

6. The tail of certain bacteriophage contracts to inject the DNA genome of the phage into the host cell. Curiously, however, neither the tail nor the baseplate proteins hydrolyze ATP or any other molecule as a source of energy. Based on this, what kind of energy likely drives tail contraction? a. b. c. d.

Heat energy produced when the phage binds to the host cell’s surface Kinetic energy generated by the tail fibers of the phage Potential energy stored in the folding state of the tail proteins Kinetic energy stored in nucleotides of the genetic material in the head

7. Phage conversion is an example of a. b. c. d.

horizontal gene transfer. vertical gene transfer. gene drift. kin selection.

SYNTHESIZE 1. E. coli lysogens derived from infection by phage λ can be induced to form progeny viruses by exposure to radiation. The inductive event is the destruction of a repressor protein that keeps the prophage genome unexpressed. What might be the normal role of the protein that recognizes and destroys the λ repressor? 2. Which is the greater threat to human health: antigenic drift in influenza viruses or antigenic shift in influenza viruses? 3. Efforts to produce an HIV vaccine have met with limited success. What aspects of the virus and its replicative strategy make it difficult to produce a vaccine against HIV? What other kind of virus might be similarly difficult to vaccinate against? What similarities and differences exist between the two types of virus that account for the difficulties in vaccine production? 4. If you could develop a drug to treat COVID-19, why might a drug that interferes with the virus’s RNA-dependent RNA polymerase be a better option than a drug that stops the virus from binding to the ACE2 receptor? 5. Assuming that the average viral density in a liter of seawater is about 108 virions, that the volume of the Pacific Ocean is about 7*1020 liters, and that the average oceanic virus has a molecular mass of 10,000 g/mol, what is the mass of virions in the Pacific Ocean? chapter

26 Viruses 559

27 CHAPTER

Prokaryotes Chapter Contents 27.1

Prokaryotic Diversity

27.2 Prokaryotic Cell Structure 27.3 Prokaryotic Genetics 27.4 The Metabolic Diversity of Prokaryotes 27.5 Microbial Ecology 27.6 Bacterial Diseases of Humans

0.6 μm David M. Phillips/Science Source

Introduction

Visual Outline Prokaryotes include Domain Bacteria Domain Archaea exhibit

Diversity

H+

H+

Cell structure Genetic information Metabolism

variation seen in

some species have

play roles in

Beneficial uses to humans

Ecosystems

via

by associating with

Nutrient cycling

Plants and animals but pathogens cause Disease

for example

Bioremediation

All cells can be characterized as one of two fundamental cell types: prokaryote or eukaryote. The term prokaryote describes a cell type, or collectively the organisms in the Domains Bacteria and Archaea. Although all replicating cells share the common characteristics of having ribosomes, cytoplasm, a plasma membrane, and DNA as their genetic material, there are important differences between eukaryotic and prokaryotic cells. Prokaryotic cells are significantly more abundant than are eukaryotic cells; estimates suggest approximately 1031 prokaryotic cells, and four to five orders of magnitude fewer eukaryotic cells in the biosphere. In the human body there are about the same number of bacterial cells as there are eukaryotic cells, but the balance can vary to favor one or the other cell type with just one bowel movement! Although some prokaryotes are important agents of potentially deadly

diseases (like the bacteria Pseudomonas aeruginosa pictured here), an overwhelmingly greater number play essential roles in nutrient cycling and energy transformations in ecosystems. Without these prokaryotes, life as we know it may not have appeared, and most definitely could not continue.​

27.1 Prokaryotic

Diversity

Learning Outcomes 1. Differentiate between archaea, bacteria, and eukarya. 2. Describe the basic characteristics of bacteria and archaea. 3. Describe classification systems for prokaryotes.

While the absolute number of prokaryotic species remains unclear, it is likely that there are many more species of bacteria and archaea than there are species of eukarya. Still, difficulties of defining “species” aside, it is not the number of species of prokaryotes versus eukaryotes that is interesting. More important is the diversity of ways prokaryotes are successful relative to eukaryotes. Prokaryotes colonize environments that would be lethal to most eukaryotic organisms. The metabolic and physiological diversity of prokaryotes has allowed their success in virtually all ecological niches ever explored.​

A brief history of microbiology Because prokaryotic cells are generally significantly smaller than eukaryotic cells, they remained hidden until the mid-17th century when Anton van Leeuwenhoek observed what we now know to be bacteria using a homemade microscope. Even before this, however, Girolamo Fracastoro suggested that disease was caused by unseen organisms. The history of microbiology entwines these two strands of improved technology for visualizing the unseen (microscopy), and investigating the nature of infectious diseases. Central to this was also the prolonged debate about the very nature of how living things arise: from other living things or by spontaneous generation from nonliving matter. The controversy over spontaneous generation (see figure 1.4) continued into the 19th century, when Louis Pasteur and others conclusively showed that life does not arise spontaneously. Ultimately, Robert Koch was able to identify and culture the causative agent for the disease anthrax. He proposed the following four postulates, Koch’s postulates, to prove a causal relationship between a microorganism and a disease: 1. The microorganism must be present in every case of the disease and absent from healthy individuals. 2. The putative causative agent must be isolated and grown in pure culture. 3. The same disease must result when the cultured microorganism is used to infect a healthy host. 4. The same microorganism must be isolated again from the diseased host. Even Koch realized that these postulates do not hold true for every case of infectious disease, however. For example, many

people infected with the bacterium causing cholera are asymptomatic, and therefore the first postulate is violated. A causal relationship between an infectious agent and a disease is better established using a set of molecular criteria, some of which bear resemblance to Koch’s original postulates. The study of microorganisms and disease also led to advances in knowledge about the immune system, and how exposure to a disease-causing agent can prevent future disease (see chapter 50). Due to our increasing knowledge about the role of prokaryotes in ecosystems, and how they can be manipulated to produce useful products, microbiology is more relevant than ever. We can now survey microbial diversity using an approach called metagenomics: DNA is sampled directly from an environment and sequenced; then, using computer algorithms to assemble genome sequences, new species can be identified. This has revealed even greater diversity of prokaryotic forms than suspected, along with novel adaptations to diverse environments. Although we recognize thousands of different kinds of prokaryotes, it is estimated that less than 10% of prokaryotic species have been identified and/or characterized. Prokaryotes are the oldest, the structurally simplest, and likely the most abundant forms of life. Prokaryotes were abundant for over a billion years before eukaryotes appeared. Early photosynthetic bacteria altered the Earth’s atmosphere by producing oxygen, which stimulated the expansion of prokaryotes into oxygenic environments and facilitated the appearance of eukaryotes capable of utilizing oxygen. Prokaryotes are ubiquitous and live everywhere eukaryotes do, but also flourish in places no eukaryote could live. Bacteria and archaea have been found in deep-sea hydrothermal vents, at volcanic rims, and deep within glaciers. Some of the extreme environments in which prokaryotes can be found would be lethal to any other life-form. Many archaea are extremophiles: they live in hotsprings (figure 27.1), in hypersaline environments that would dehydrate other cells, and in atmospheres rich in toxic gases such as hydrogen sulfide. Some of these harsh environments may be similar to the conditions present on early Earth when life first appeared. It is possible that prokaryotes became adapted to survive

Figure 27.1 Hotsprings such as these in Yellowstone National Park contain thermophilic prokaryotes. Robert Glusic/Getty Images

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27 Prokaryotes 561

these harsh conditions early on and have retained the ability to exploit these areas as the rest of the atmosphere has changed.​

Prokaryotes are fundamentally different from eukaryotes Prokaryotes differ from eukaryotes in many important ways. These differences represent some of the most fundamental distinctions that separate any groups of organisms: Unicellularity. With a few exceptions, prokaryotes are singlecelled. In some species, individual cells adhere to one another and form filaments or aggregates; however, the cells retain their individuality. Even in these cases, the characteristics of the organism are the characteristics of the individual cells, unlike in a multicellular organism. In their natural environments, many prokaryotes appear to be capable of forming complex communities of different species called biofilms (figure 27.2). These associations are more resistant to antibiotics, desiccation, and other environmental stressors than is a simple colony of a single type of microbe, such as might grow on an agar plate in the lab. Cell size. As new species of prokaryotes are discovered, investigators are finding that the size of prokaryotic cells varies by as much as 100,000 fold. Thiomargerita namibiensis is the largest known bacteria, with cells up to 0.75 mm in diameter—just visible to the naked eye (figure 27.3). Most prokaryotic cells, however, are 1 μm or less in diameter, whereas most eukaryotic cells are about 10 times larger. While this is a useful generalization, there are very small eukaryotic cells as well as very large prokaryotes. Nucleoid. Prokaryotes lack a membrane-bound nucleus; instead they usually have a single circular chromosome made up of DNA and histone-like proteins in a nucleoid region of the cytoplasm. The exceptions to this are Vibrio cholerae, which has two circular chromosomes, and Borrelia species, which have multiple linear chromosomes. Prokaryotic cells also often have smaller circular DNA molecules called plasmids, which usually confer some selective advantage, but are not essential. Cell division and genetic recombination. Cell reproduction in eukaryotes takes place by mitotic or meiotic cell division and uses spindles made up of microtubules to segregate chromosomes. Cell division in prokaryotes takes place

Figure 27.3 Cells of the bacterium Thiomargarita namibiensis can be as large as 0.75 mm across. mainly by binary fission (see chapter 10), which does not use a spindle. Prokaryotes also do not have a sexual cycle, although they do exchange genetic material extensively. These mechanisms are collectively called horizontal gene transfer; while not related to reproduction, they play important roles in evolutionary change. Internal compartmentalization. Prokaryotes do not have extensive membrane-bound organelles like eukaryotes; however, some species have structures that can isolate specific metabolic pathways, or provide storage. These structures may be bounded by a lipid bilayer or monolayer, or by a protein shell. The plasma membrane can also be extensively invaginated to isolate processes like cellular respiration or photosynthesis. Flagella. Prokaryotic flagella are composed of a single protein fiber and not the 9 + 2 arrangement of microtubules found in eukaryotic flagella. Bacterial flagella have a twist, are rigid, and rotate to drive cell movement; eukaryotic flagella, on the other hand, have a whiplike motion (described in more detail in section 27.2 and in figure 27.9). Pili. Some species of bacteria have numerous filaments of protein, extending from their surface called pili or fimbriae. These are structurally diverse and may be involved in the exchange of genetic material, in the attachment of pathogenic species to host tissues, and occasionally in motility. Metabolic diversity. The metabolic diversity of prokaryotes reflects the diversity of environments in which they can live. Different prokaryotes can exploit a variety of organic and inorganic electron donors and acceptors to perform cellular respirations, can ferment a variety of organic substances, and can use a variety of different carbon sources (see section 27.4). In these respects, there is significantly greater metabolic and nutritional diversity amongst the prokarya than amongst the eukarya. Some bacteria are photosynthetic and may produce oxygen or some other oxidized by-product.​

Despite similarities, bacteria and archaea differ 750×

Figure 27.2 A biofilm of the bacteria found in dental plaque on a toothbrush bristle. Steve Gschmeissner/Science Source 562 part V Diversity of Life on Earth

Archaea and bacteria are similar in that both have a prokaryotic cellular structure and are ancient forms of life; however, they vary considerably at the biochemical and molecular levels. Moreover, phylogenies based on rRNA sequences show that the archaea are

more closely related to the eukarya than they are to the bacteria (see figure 27.5). They differ in four key ways: plasma membrane structure, cell wall structure, DNA replication, and gene expression. There are also some metabolic differences between the two domains. Plasma membranes. All prokaryotes have plasma membranes with a fluid mosaic structure (see chapter 5). The plasma membranes of archaea differ from those of both bacteria and eukaryotes. Archaeal membrane lipids are composed of glycerol linked to hydrocarbon chains by ether linkages, not the ester linkages seen in bacteria and eukaryotes (figure 27.4a). These hydrocarbons may also be branched, and they may be organized as tetraethers that form a monolayer instead of a bilayer (figure 27.4b). In the case of some hyperthermophiles, the majority of the membrane may be this tetraether monolayer. This structural feature is part of what allows these archaea to withstand high temperatures. Cell wall. Most bacteria and archaea have cell walls that confer shape and rigidity upon the cell and prevent cell lysis due to high osmotic pressures. The cell walls of bacteria contain varying amounts of peptidoglycan, a molecule formed from carbohydrate polymers linked together by peptide crossbridges. The peptide cross-bridges also contain D-amino acids, which are never found in proteins produced by translation. The cell walls of archaea lack peptidoglycan, although some have pseudomurein, which is similar to peptidoglycan in structure and function. This wall layer is also a carbohydrate polymer with peptide cross-bridges, but the carbohydrates are different, and the peptide cross-bridge structure also differs. Other archaeal cell walls have been found to be composed of a variety of proteins and carbohydrates, making generalizations difficult. DNA replication. Although both archaea and bacteria have a single replication origin per chromosome, the nature of this origin and the proteins that act there are quite different. Archaeal initiation of DNA replication is more similar to that of eukaryotes (see chapter 14). Gene expression. Archaea has a single RNA polymerase, which more closely resembles the eukaryotic RNA polymerases Bacterial or Eukaryotic Lipid

Glycerol

than they do the single bacterial RNA polymerase. Some of the translation machinery of archaea is also more similar to that of eukaryotes than to that of bacteria. (see chapter 15).

Classifying prokaryotes Prokaryotes are not easily classified according to their forms, and only recently has enough been learned about their biochemical and metabolic characteristics to develop a satisfactory overall classification scheme comparable to that used for other organisms. With no clear definition of what constitutes a prokaryotic species, systematics has been difficult.​

Early classification characteristics Early taxonomies for classifying prokaryotes relied on readily observable or microscopically detectable differences between organisms or groups of organisms. Key characteristics initially used to classify prokaryotes were: 1. photosynthetic ability 2. cell wall structure 3. motility 4. unicellular or colony-forming or filamentous 5. spore-forming ability 6. pathogenicity

Molecular approaches to classification As technologies have advanced, combinations of approaches have been used to create taxonomies. For example, information from genomics can be combined with phenotypic and phylogenetic data to create more meaningful and well-informed classifications. Molecular approaches include the following: 1. the analysis of the amino acid sequences of key proteins 2. the analysis of nucleic acid–base sequences by establishing the percent of guanine and cytosine 3. nucleic acid hybridization, which is essentially the mixing of single-stranded DNA from two species and

Archaeal Lipid

Tetraether

CH2

CH

CH2

CH2

CH2

CH2

CH2

CH

CH2

OH

O

O

OH

O

O

OH

O

O

C

C

O

O

OH

CH2

CH

CH2

C

O

C

O

Monolayer Ester bond

a.

Ether bond

b.

Figure 27.4 Archaeal membrane lipids. a. Archaeal membrane lipids are made using a glycerol skeleton similar to that of bacterial and

eukaryotic lipids, but the hydrocarbon chains are connected to the glycerol by ether linkages, not ester linkages. The hydrocarbons can also be branched and even contain rings. b. These lipids can also form as tetraethers instead of diethers. The tetraether forms a monolayer, as it includes two polar regions connected by hydrophobic hydrocarbons. chapter

27 Prokaryotes 563

determination of the amount of base-pairing (closely related species will have more bases pairing) 4. gene and RNA sequencing, especially looking at ribosomal DNA sequences (rDNA) 5. whole-genome sequencing

Domain Bacteria

The three-domain system of phylogeny, originated by Carl Woese (figure 27.5 and chapter 25), relies on all of these molecular methods, but emphasizes the comparison of rDNA sequences to establish the evolutionary relatedness of all organisms. The rDNA sequences were chosen for their high degree of evolutionary conservation to ask questions about these most ancient splits in the tree of life. Based on these sorts of molecular data, several groupings of prokaryotes have been proposed. The most widely accepted is that presented in Bergey’s Manual of Systematic Bacteriology, second edition, most recently published in 2012 (figure 27.6). At the same

Euryarchaeota

Aquificae

Domain Archaea

Domain Eukarya

Common ancestor

Figure 27.5 The three domains of life. The two prokaryotic domains, Archaea and Bacteria, are not closely related, although both are prokaryotes. In many ways (see text), archaea resemble eukaryotes more closely than they resemble bacteria. This phylogeny is based on rRNA sequences.

Bacilli

Spirochetes

Actinobacteria

1 μm 1.37 μm Archaea differ greatly from bacteria. Although both are prokaryotes, archaeal cell walls lack peptidoglycan; plasma membranes are made of different kinds of lipids than bacterial plasma membranes; RNA and ribosomal proteins are more like those of eukaryotes than those of bacteria. Mostly anaerobic. Examples include Methanococcus, Thermoproteus, Halobacterium.

The Aquificae represent the deepest or oldest branch of bacteria. Aquifex pyrophilus is a rod-shaped hyperthermophile with a temperature optimum at 85°C; a chemoautotroph, it oxidizes hydrogen or sulfur. Several other related phyla are also thermophiles.

4 μm Gram-positive bacteria. Largely solitary; many form endospores. Responsible for many significant human diseases, including anthrax (Bacillus anthracis); botulism (Clostridium botulinum); and other common diseases (Staphylococcus, Streptococcus).

22 μm Some gram-positive bacteria form branching filaments and some produce spores; often mistaken for fungi. Produce many commonly used antibiotics, including streptomycin and tetracycline. One of the most common types of soil bacteria; also common in dental plaque. Streptomyces, Actinomyces.

Thermophiles Crenarchaeota

Euryarchaeota

Aquificae

Thermotogae

Chloroflexi

564 part V Diversity of Life on Earth

Bacteria

Long, coil-shaped cells that stain gramnegative. Common in aquatic environments. Rotation of internal filaments produces a corkscrew movement. Some spirochetes such as Treponema pallidum (syphilis) and Borrelia burgdorferi (Lyme disease) are significant human pathogens.

Gram-positive bacteria DeinococcusThermus

Low G/C (Firmicutes) Bacilli

Archaea

3 μm

Clostridium

High G/C Actinobacteria

time, large-scale sequencing of randomly sampled collections of bacteria shows an incredible amount of diversity. These new data indicate that the vast majority of bacteria have never been cultured and studied in any detail.

27.2 Prokaryotic Learning Outcomes

Learning Outcomes Review 27.1

1. Describe the general features common to prokaryotic cells. 2. Explain why bacteria can stain gram-positive or gram-negative. 3. Describe features that might discriminate one prokaryote from another.

The study of prokaryotic organisms was first made possible by microscopes. Early microbiology grew from the study of disease-causing agents. Prokaryotes lack both a membranebound nucleus and the organelles seen in eukaryotes, and they reproduce by binary fission. Although they are similar in some respects, bacteria and archaea have important structural and physiological differences. Prokaryotic classification has been based on physical characteristics, but is now informed by molecular biology and genomics. A vast number of prokaryotes have not been studied because they have not been cultured. ■■

Prokaryotic cells are relatively simple, but they can be categorized based on morphology. They also have some structural variations that allow them to be differentiated with stains.​

Prokaryotes are diverse in shape and size

What features distinguish archaea from bacteria?

Cyanobacteria

Cell Structure

Beta

Although it is an oversimplification, it is useful to classify bacteria based on obvious morphologies. Most prokaryotes exhibit one of

Gamma

Figure 27.6 Some major clades of prokaryotes. The

Delta

classification adopted here is that of Bergey’s Manual of Systematic Bacteriology, second edition, 2001. G/C refers to %G/C in genome. 

25 μm

10 μm Cyanobacteria are a form of photosynthetic bacterium common in both marine and freshwater environments. Deeply pigmented; often responsible for “blooms” in polluted waters. Both colonial and solitary forms are common. Some filamentous forms have cells specialized for nitrogen fixation.

0.5 μm A nutritionally diverse group that includes soil bacteria like the lithotroph Nitrosomonas that recycle nitrogen within ecosystems by oxidizing the ammonium ion (NH4+). Other members are heterotrophs and photoheterotrophs.

Gammas are a diverse group including photosynthetic sulfur bacteria, pathogens, like Legionella, and the enteric bacteria that inhabit animal intestines. Enterics include Escherichia coli, Salmonella (food poisoning), and Vibrio cholerae (cholera). Pseudomonas are a common form of soil bacteria, responsible for many plant diseases, and are important opportunistic pathogens.

Photosynthetic Spirochetes

Cyanobacteria

Chlorobi

750 μm The cells of myxobacteria exhibit gliding motility by secreting slimy polysaccharides over which masses of cells glide; when the soil dries out, cells aggregate to form upright multicellular colonies called fruiting bodies. Other delta bacteria are solitary predators that attack other bacteria (Bdellovibrio) and bacteria used in bioremediation (Geobacter).

SPL/Science Source; Prof. Dr. R. Rachel and Prof. Or. K. O. Stetter, University of Regensburg, Regensburg, Germany; Andrew Syred/Science Source; Microfield Scientific Ltd/Science Source; Alfred Pasieka/Science Source2; Don Rubbelke/ McGraw Hill; Dennis Kunkel Microscopy/ Science Source; Prof. Dr. Hans Reichenbach, Helmholtz Centre for Infection Research, Braunschweig

Proteobacteria Beta

Gamma

Alpha– Rickettsia

Epsilon– Helicobacter

Delta

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27 Prokaryotes 565

three basic shapes: rod-shaped, often called a bacillus (plural, bacilli); coccus (plural, cocci), spherical or oval-shaped; and spirillum (plural, spirilla), long and helix-shaped. Spirochetes appear similar to spirilla; however, they tend to be narrower with a looser twist. Some types of prokaryote have no specific shape and are said to be pleiomorphic. Bacillus

Coccus

0.5 μm

Spirillum

2 μm

3 μm

Dr. Gary Gaugler/SPL/Science Source; CNRI/Science Source; Centers for Disease Control and Prevention

The bacterial cell wall is the single most important contributor to cell shape. Bacteria that normally lack cell walls, such as the mycoplasmas, do not have a set shape. Some rodshaped and spherical bacteria form colonies, adhering end-toend after they have divided, forming chains. Some bacterial cells change into stalked structures or grow long, branched filaments. ​

Prokaryotes are generally smaller than eukaryotic cells The range of cell morphologies seen in prokaryotes makes it difficult to describe cell size; however, a reasonable size range is from 200 nm to 0.7 mm. The average bacillus is anywhere from 0.5 µm to 3 µm in diameter and up to 15 µm long. The average eukaryotic cell is about 20 to 25 µm in diameter, but again, there are significant variations. More important than absolute size ranges are the consequences of being small. A coccoid-shaped bacteria with a cell diameter of 2 µm has a surface area-to-volume ratio of 3. As the cell diameter increases, the surface area-to-volume ratio drops rapidly. A simple doubling of the diameter results in a halving of the surface areato-volume ratio. This ratio is important because nutrients are taken up across the surface of cells. The more surface area a cell has in proportion to the amount of volume (containing metabolically demanding cytoplasm), the more rapidly it can take up nutrients. For a cell to divide, the cell must grow to a certain size and replicate its chromosome(s), processes requiring nutrient uptake. Prokaryotic cells that are smaller than eukaryotic cells can rapidly absorb nutrients, grow rapidly, and quickly replicate their chromosomes. Every time a chromosome replicates, there is a chance for mutations. A rapidly dividing prokaryote can accumulate mutations that it can test in its environment for a selective advantage. Subsequently, small cells evolve

566 part V Diversity of Life on Earth

more quickly than do their larger brethren. This is particularly problematic when bacteria can test out mutations in a few generations to evade antibiotics that take us years to develop and test!

​ ost prokaryotes have cell walls and other M external structures Most, but not all, prokaryotes possess a cell wall and some number of other external structures. A cell wall, if present, is often complex, consisting of many layers. Minimally, it consists of peptidoglycan, a polymer composed of a rigid network of polysaccharide strands cross-linked by peptide side chains. The cell wall maintains the shape of the cell and protects the cell from swelling and rupturing in hypotonic solutions, which are commonly found in the environment. The archaea do not possess peptidoglycan, but some have a similar structure called pseudomurein. In bacteria, the structure of the cell wall can be used as the basis of creating two groups: the gram-positive and the gram-­negative bacteria.​

Gram-positive and gram-negative bacteria Two types of bacteria can be identified using a staining process called the Gram stain. Gram-positive bacteria have a thicker peptidoglycan wall and stain purple, whereas the more common gram-negative bacteria contain less peptidoglycan and do not retain the purple-colored dye. These gram-negative bacteria can be stained with a red counterstain and then appear dark pink (figure 27.7). In the gram-positive bacteria, the peptidoglycan forms a thick, complex network around the outer surface of the cell. This network also contains lipoteichoic and teichoic acid, which protrudes from the cell wall. In the gram-negative bacteria, a thin layer of peptidoglycan is sandwiched between the plasma membranes and a second outer membrane (figure 27.8). The outer membrane contains large molecules of lipopolysaccharide, lipids with polysaccharide chains attached. The outer membrane layer makes gram-negative bacteria resistant to many antibiotics that interfere with cell-wall synthesis in gram-positive bacteria.​

S-layers In some bacteria and archaea, an additional protein or glycoprotein layer forms a rigid paracrystalline surface called an S-layer outside of the peptidoglycan or outer membrane layers of gram-positive and gram-negative bacteria, respectively. Among the archaea, the S-layer is almost universal and can be found outside of a pseudomurein layer or, in contrast to the bacteria, may be the only rigid layer surrounding the cell. The functions of S-layers are diverse and variable; they can attach the cell to a surface, afford protection to the cell, trap molecules close to the cell surface, or act as a selectively permeable structure.​

1. Crystal violet is applied.

Gram-positive

2. Gram’s iodine is applied.

Gram-negative

Both cell walls affix the dye.

Gram-positive

3. Alcohol wash is applied.

Gram-negative

Crystal violet–iodine complex formed inside cells. All one color.

4. Safranin (red dye) is applied.

Gram-positive

Gram-negative

Gram-positive

Gram-negative

Alcohol dehydrates thick PG layer trapping dye complex.

Alcohol has minimal effect on thin PG layer.

Dark purple masks the red dye.

Red dye stains the colorless cell.

a.

Figure 27.7 The Gram stain. a. The thick peptidoglycan (PG) layer encasing

gram-positive bacteria traps crystal violet dye, so the bacteria appear purple in a gramstained smear (named after Hans Christian Gram—Danish bacteriologist, 1853–1938, who developed the technique). Because gram-negative bacteria have much less peptidoglycan (located between the plasma membrane and an outer membrane), they do not retain the crystal violet dye and so exhibit the red counterstain (usually a safranin dye). b. A micrograph showing the results of a Gram stain with both gram-positive and gramnegative cells. (b) CDC/Dr. William A. Clark

b.

Gram-positive

10 μm

Gram-negative Lipoteichoic acid

Lipopolysaccharide

Porin Protein

Protein Teichoic acid

Cell interior

Cell interior

Plasma membrane

Cell wall (peptidoglycan)

Plasma membrane

Peptidoglycan

Outer membrane

Cell wall

Figure 27.8 The structure of gram-positive and gram-negative cell walls. The gram-positive cell wall (left) is much simpler than the gram-negative cell wall. The gram-positive cell wall is composed of a single, thick layer of peptidoglycan; molecules of lipoteichoic acid and teichoic acid are embedded in the wall and exposed on the surface of the cell. The gram-negative cell wall is composed of multiple layers. The peptidoglycan layer is thinner than in gram-positive bacteria and is surrounded by an additional membrane composed of lipopolysaccharide. Porin proteins form aqueous pores in the outer membrane. The space between the outer membrane and the peptidoglycan is called the periplasmic space.

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27 Prokaryotes 567

Capsules and slime layers In some bacteria, an additional gelatinous layer, the capsule, surrounds the other wall layers and can be detected relatively easily by staining and microscopy. A more loosely organized form of capsule that is harder to detect is called a slime layer. A capsule enables a prokaryotic cell to adhere to surfaces and to other cells, and, most important, to evade an immune response by interfering with recognition by phagocytic cells. Therefore, a capsule often contributes to the ability of bacteria to cause disease.​

Flagella and pili Some prokaryotes are motile. Many prokaryotic cells possess one flagellum at one end, some possess one at each end of the cell, and some possess many flagella in clusters or distributed around the cell. While appearing similar, these structures evolved independently in bacteria and archaea. The central filament of the bacterial flagella can be from 0.3 to 12 μm long and 10 to 20 nm wide, and is made from a single protein called flagellin. The analogous structure in archaea is called an archaellum; it has a central filament composed of a different protein, related to the proteins found in bacterial pili. The structures anchoring flagella to the membrane and cell wall are also different (figure 27.9). Cell motility is due to rotation of the protein fiber, but this is powered by different energy sources: a proton gradient in bacteria and ATP in archaea. Pili (singular, pilus) are numerous fine structures that occur on the cells of some gram-negative prokaryotes. They are shorter

than prokaryotic flagella and only about 7.5 to 10 nm thick. Pili can be used to attach a cell to a surface, and may be important in allowing colonization of tissues by pathogens. Some pili are adapted to be involved in the sharing of genetic information, and other specialized pili may also be used to cause a cell to “twitch,” which can result in directional movement.​

Endospores Some prokaryotes are able to form endospores: a thick wall develops around their genome and a small amount of cytoplasm when they are exposed to environmental stress. These endospores are highly resistant to environmental stress such as heat, and when environmental conditions improve, they can germinate and return to normal cell division to form vegetative cells after decades or even centuries of dormancy. The bacteria that cause tetanus, botulism, and anthrax are all capable of forming spores. With a puncture wound, tetanus endospores may be driven deep into the skin, where conditions are favorable for them to germinate and cause disease, or even death. ​

The internal organization of prokaryotes Relative to eukaryotic cells, prokaryotes have a relatively simple interior organization. They lack the extensive functional compartmentalization seen within eukaryotic cells, but they do have the following structures: Membrane folds. Many prokaryotes possess invaginated regions of the plasma membrane that function in respiration or photosynthesis (figure 27.10). The folded plasma

Hook Filament

Outer membrane Peptidoglycan portion of cell wall Outer protein ring Inner protein ring

a.

H+

H+

Plasma membrane

b.

20 nm

Figure 27.9 The flagellar motor of a gram-negative bacterium. a. A protein filament, composed of the protein flagellin,

is attached to a protein rod that passes through a sleeve in the outer membrane and through a hole in the peptidoglycan layer to rings of protein anchored in the cell wall and plasma membrane, like rings of ball bearings. The rod rotates when the inner protein ring attached to the rod turns with respect to the outer ring fixed to the cell wall. The inner ring is an H+ ion channel, a proton pump that uses the flow of protons into the cell to power the movement of the inner ring past the outer one. b. Electron micrograph of a bacterial flagellum. 

(Right) Julius Adler

568 part V Diversity of Life on Earth

a.

0.5 μm

b.

0.85 μm

Figure 27.10 Prokaryotic cells often have complex internal membranes. a. This aerobic bacterium exhibits extensive

respiratory membranes (long dark curves that hug the cell wall) within its cytoplasm, not unlike those of mitochondrial cristae. b. This cyanobacterium has thylakoid-like membranes (ripplelike shapes along the edges and in the center) that provide a site for photosynthesis.  (Left) Dr. W.J. Ingledew/Science Source; (Right) Biophoto Associates/Science Source

membrane provides an increased amount of surface area for holding respiratory or photosynthetic proteins and subsequently increase metabolic efficiency. The folding strategy seen in prokaryotic cells is fundamentally the same as that seen in eukaryotic organelles that have additional membranes (chloroplasts) or folded membranes (mitochondria) to increase surface area. The nucleoid region. Prokaryotic cells do not package their chromosomes in a membrane-bounded nucleus. Instead, their circular DNA chromosomes are condensed to form a visible region of the cell known as the nucleoid region. Many prokaryotic cells also possess plasmids, which are small, extrachromosomal, independently replicating circles of DNA. Plasmids contain only a few genes, and although these genes may confer a selective advantage, they are not essential for the cell’s survival. Ribosomes. Prokaryotic ribosomes are smaller than those of eukaryotes and differ in protein and RNA content. Antibiotics such as tetracycline and chloramphenicol can tell the difference, however; they bind to prokaryotic ribosomes and block protein synthesis, but they do not bind to eukaryotic ribosomes. Internal compartments. While neither bacteria nor archaea have consistent internal compartments found in all cells, they can have both lipid- and protein-bounded compartments. A small number of membrane-bounded structures have been characterized, such as the magnetosome in magnetotactic bacteria. More common are protein shells that are called bacterial microcompartments (BMC), which isolate specific metabolic processes to either increase concentration of reactants, or protect the cell from toxic metabolic intermediates. For example, in cyanobacteria, carboxysomes contain the enzyme RUBISCO, and increase the local CO2 concentration, which increases the efficiency of carbon fixation (chapter 8). Excess proteins can also result in the formation of inclusion bodies, and shells of either lipids or protein can be used for storage of specific metabolites.

Learning Outcomes Review 27.2

The three basic shapes of prokaryotes are rod-shaped, spherical, and spiral-shaped. Bacteria have a cell wall containing peptidoglycan, which is the basis for the Gram stain. Gram-positive bacteria have a thick cell wall, relative to gram-negative species. Many also have an external capsule. Some bacteria have flagella and pili. Some can form heat-resistant endospores. Bacteria may also have membrane- or protein-bound internal compartments to isolate metabolic processes or store materials; some biochemical processes are performed on extensive infoldings of the plasma membrane. Prokaryotic DNA is localized in a nucleoid region. ■■

If you performed a Gram stain on a gram-positive species but forgot to treat the cells with the iodine, how would they appear through a microscope?​

27.3 Prokaryotic

Genetics

Learning Outcomes 1. Compare and contrast the mechanisms of DNA exchange in prokaryotes. 2. Describe how genes are mapped in E. coli. 3. Describe how antibiotic resistance can spread in and between bacterial populations.

In sexually reproducing populations, traits can only be transferred vertically, from parent to child. Prokaryotes do not reproduce sexually; however, they can exchange DNA between different cells of the same species and in many cases, between cells of different species. This horizontal gene transfer occurs when genes move from one cell to another by conjugation, requiring cell-to-cell contact, or by means of viruses (transduction). Some species of bacteria can also pick up genetic material directly from the environment (transformation). All of these processes have been observed in archaea, but the study of archaeal genetics is hampered by the difficulty of culturing most species. We concentrate here on bacterial processes, primarily those of E. coli, which has been studied extensively. ​

Transformation is the uptake of DNA from the environment Natural transformation occurs in some gram-negative and some gram-positive species, which are said to be “competent.” Transformation occurs when one bacterial cell has died and ruptured, spilling its fragmented chromosomal DNA, or plasmids, into the environment. This DNA can be absorbed by another cell and incorporated into its genome, thereby “transforming” it (figure 27.11). The cell taking up the DNA is said to be “transformed” because it likely obtains a new characteristic after transformation. Some species of gram-positive and gram-negative bacteria are competent for transformation, although the mechanisms seem to differ between the groups. The proteins involved in the process of natural transformation are encoded by the bacterial chromosome. The implication is that natural transformation may be the only one of the mechanisms of DNA exchange that evolved as part of the normal cellular machinery. The transfer of chromosomal DNA by either conjugation or transduction—which we discuss next— can be thought of as accidents of plasmid or phage biology, respectively. Transformation is also important in molecular cloning, which relies heavily on the bacterium E. coli; however, E. coli is not naturally competent. When transformation is done in the laboratory, it is called artificial transformation. Artificial

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27 Prokaryotes 569

Cell death of a bacterium causes release of DNA fragments.

A DNA fragment is taken up by another live cell.

DNA is incorporated by homologous recombination.

Cell contains DNA from dead donor cell.

Figure 27.11 Natural transformation. Natural transformation occurs when a cell dies and releases its contents to the surrounding environment. Genomic DNA is usually fragmented, and small pieces can be taken up by other, living cells. The DNA taken up can replace chromosomal DNA by homologous recombination as in conjugation and transduction. If the new DNA contains different alleles from the chromosome of the recipient cell, the phenotype of the cell changes, possibly providing a selective advantage.

transformation is useful for cloning and DNA manipulation (see chapter 17).​

Transduction is the transfer of DNA between prokaryotes​by viruses Horizontal transfer of DNA can also be mediated by viruses that infect prokaryotes. In generalized transduction, virtually any gene can be transferred between cells; in specialized transduction, only specific genes can be transferred.​

Generalized transduction Generalized transduction can be thought of as an accident of the biology of some types of lytic phage (see chapter 26). In these viruses, after the viral genome is replicated and the phage head is constructed, the phage packaging machinery puts DNA into the phage head until no more fits. Sometimes the phage begins with bacterial DNA instead of phage DNA and packages this DNA into a phage head accidentally (figure 27.12). When this viral particle goes on to infect another cell, it injects into the host cell bacterial DNA instead of viral DNA. This DNA can then be incorporated into the host cell’s chromosome by homologous recombination. Generalized transduction has also been used to map genes in the genome of E. coli. In generalized transduction, the closer together two genes are on the host cell chromosome, the more likely it is that they will be transferred in a single transduction event. This can be expressed mathematically as the cotransduction frequency. If this frequency is combined with information obtained from experiments exploiting conjugation (see below), distances between genes can be determined with some accuracy.​

Specialized transduction Specialized transduction is limited to phage that exhibit a lysogenic life cycle (see chapter 26). A good example of such a phage is phage λ from E. coli. When λ infects a cell and its genome 570 part V Diversity of Life on Earth

integrates into the host chromosome, it does not destroy the cell but is passed on by cell division. In this lysogenic state, the phage is called a prophage and it is dormant. The prophage encodes the functions necessary to eventually excise itself and undergo lytic growth, leading to the death of the cell. If this excision event is imprecise, the prophage may take some chromosomal DNA with it; when the phage DNA plus the chromosomal DNA are packaged into a virion, a specialized transducing phage is created. Because the phage head can carry only as much DNA as is found in the phage genome, imprecise excision of the prophage results in deletion of phage genes. Thus, specialized transducing phage may be defective if genes necessary for phage replication are deleted during packaging. Upon infection of an appropriate host by a specialized transducing phage, the DNA in the phage head can be integrated into the host chromosome. Phage particles with DNA that can integrate as prophages may become trapped in the host genome if the genes necessary for excision and viral replication become defective by mutation or were lost during the creation of the transducing particle. The E. coli genome contains a number of such cryptic prophage, some of which encode functions important to the cell and must now be considered part of the host genome.​

Conjugation depends on the presence of a conjugative plasmid Plasmids encode functions that are not necessary to the organism but that provide a selective advantage in particular environments. For example, antibiotic resistance is not necessary, but is obviously advantageous in the presence of the antibiotic. Some plasmids can be transferred from one cell to another via conjugation. The bestknown transmissible plasmid is the E. coli F plasmid (fertility factor). Cells containing F plasmids are termed F+, or donor, cells, and cells that lack the F plasmid are F−, or recipient, cells. The F plasmid is an extrachromosomal piece of DNA that uses cellular machinery for replication. ​

Infection with Phage

Phage adheres to cell.

Phage DNA is injected into cell.

Figure 27.12 Transduction by generalized transducing phage.  When some phage infect cells, they

trigger degradation of the host DNA into pieces. When the phage package their DNA, they can package host DNA in place of phage DNA to produce a transducing phage, as shown on the top. When a transducing phage infects a cell, it injects host DNA that can then be integrated into the host genome by homologous recombination. With a linear piece of DNA, it requires two recombination events, which replace the chromosomal DNA with the transducing DNA, as shown on the bottom. If the new allele is different from the old, the cell’s phenotype will change.

F plasmid transfer Phage DNA is replicated and host DNA is degraded.

Phage particles are packaged with DNA and are released.

Infection with Transducing Phage

Transducing phage adheres to cell.

Phage injects a piece of chromosomal DNA.

DNA is incorporated by homologous recombination.

Cell contains DNA from donor.

The F plasmid contains a DNA replication origin and several genes that promote its transfer to other cells. These genes encode protein subunits that assemble on the surface of the bacterial cell, forming a hollow pilus that is necessary for the transfer process (figure 27.13a). Once the pilus—now called a conjugation bridge—has formed between the recipient and donor cells, the F plasmid binds to a site on the interior of the donor cell just underneath the bridge. Then, by a process called rolling-circle replication, the F plasmid begins to copy its DNA. As it is replicated, the displaced single strand of the plasmid passes through the conjugation bridge into the other cell. There, a complementary strand is replicated, creating a new, double-stranded F plasmid (figure 27.13b). The recipient F− cell has acquired the characteristic of being a donor F+ cell and the F plasmid genes have spread in the population.​

Recombination between the F plasmid and the host chromosome After being transferred from a donor to a recipient cell, the F plasmid can integrate into the recipient cell’s genome by recombination. There are regions of homology with the E. coli chromosome called insertion sequences (IS), and recombination between the insertion sequences and the chromosome integrates the F-plasmid into the chromosome (figure 27.14). A cell with an integrated F plasmid is called an Hfr cell— for “high frequency of recombination” because of the high frequency with which genes from the Hfr donor cell can recombine with genes in the recipient cell. F plasmid transfer from an Hfr cell to an F− cell usually transfers only a portion of the chromosome, and not all of the F plasmid. This is because the origin of transfer is in the middle of the integrated plasmid, which would require transfer of the entire chromosome to also transfer all of the F plasmid. This transfer would take around 100 minutes, and the conjugation bridge is usually broken before transfer is completed. This leads to transfer of portions of donor chromosome that can replace regions of the recipient chromosome by two recombination events between the donor fragment and the chromosome. chapter

27 Prokaryotes 571

Conjugation bridge

Bacterial chromosome F plasmid

F+ (donor cell)

a.

F– (recipient cell)

Rolling-circle replication: single strand enters recipient cell

Second strand synthesis begins

F+

F+

b.

2 μm

Figure 27.13 Conjugation bridge and transfer of F plasmid between F+ and F― cell. a. The electron micrograph shows two E.

coli cells undergoing conjugation. The connection between the cells is the extended F pilus. b. F− cells are converted to F+ cells by the transfer of the F plasmid. The cells are joined by a conjugation bridge and the plasmid is replicated in the donor cell, displacing one parental strand. The displaced strand is transferred to the recipient cell, then replicated. After successful transfer, the recipient cell becomes an F+ cell capable of expressing genes for the F pilus and acting as a donor.  (Left) Dennis Kunkel Microscopy/Science Source

Data analysis If the excision of an F plasmid is not

precise, and some E. coli DNA is added to the F plasmid, what are the consequences when this plasmid is transferred to a new cell?

Excision

Integration

IS F plasmid

Hfr cell

Integrated F plasmid

F+ cell

Origin of transfer

Hfr cell

F+ cell

E. coli chromosome

Figure 27.14 Integration and excision of F plasmid. The

F plasmid contains short insertion sequences (IS) that also exist in the chromosome. This allows the plasmid to pair with the chromosome, and a single recombination event between two circles leads to a larger circle. This integrates the plasmid into the chromosome, creating an Hfr cell, as shown on the left. The process is reversible because the IS elements in the integrated plasmid can pair, and now a recombination event will return the two circles and convert the Hfr back to an F+ cell, as shown on the right.

572 part V Diversity of Life on Earth

Geneticists have used this process to map the order of genes on the E. coli chromosome. The transfer of genes is progressive, and the farther from the origin of transfer, the later a region will be transferred. If the process of conjugation is experimentally interrupted at specific time points, the gene order can be mapped based on time of entry (figure 27.15). Genes can be followed by using a donor with wild-type alleles that replace mutant alleles in the recipient. These experiments have shown that the genetic map of E. coli is circular, with minutes as the units of the map, and the entire chromosome is 100 minutes long. An integrated F plasmid can also excise itself by reversing the integration process (see figure 27.14). If excision is inaccurate, the F plasmid can pick up some chromosomal DNA in the process to create an F′ plasmid. Normal transfer of this F′ will produce a cell that is diploid for the material carried by the plasmid.​

Antibiotic resistance can be acquired on resistance plasmids Some conjugative plasmids can acquire antibiotic resistance genes, becoming resistance plasmids, or R plasmids. The rapid transfer of newly acquired, antibiotic resistance genes by plasmids has been an important factor in the appearance of resistant strains of the pathogenic bacteria. In some cases bacteria can become resistant to multiple antibiotics. Resistance plasmids often acquire antibiotic resistance genes through transposable elements (see chapter 18). These elements can move from chromosome to chromosome or from plasmid to chromosome and back again, and they can transfer antibiotic resistance genes in the process. If a conjugative plasmid acquires these genes, then the bacterium carrying it has a selective advantage in the presence of those antibiotics.

SCIENTIFIC THINKING Hypothesis: Conjugation using Hfr strains involves the linear transfer of information from donor to recipient cell. Prediction: If there is a linear transfer of information, then different marker genes should appear in the recipient cell at specific times following the start of conjugation. Test: Mating strains are agitated at specific time points to break the conjugation bridge, and are then plated to determine genotype. Donor Hfr azi r ton r lac+ gal +

Sample placed in blender.

Sample plated.

Mutation can lead to rapid genetic adaptation

Recipient F − azi s ton s lac − gal − Mating interrupted by agitation in blender

Percentage of cells with Hfr genetic markers

Data from Interrupted Mating

100

azi r

80

ton r

90% 85%

60 lac +

40

0

40%

gal +

20 0

10

20

30

20%

40

50

Minutes prior to interruption of conjugation

Genetic Map azi ton

0

5

10

lac gal

15

many pathogenic bacteria, including the organisms that cause dysentery, typhoid, and other major diseases. At times, some of the genetic material from these pathogenic species is exchanged with or transferred to E. coli by transmissible plasmids or bacteriophage. Because of its abundance in the human digestive tract, E. coli poses a special threat if it acquires harmful traits, as evidenced by outbreaks of the food-borne O157:H7 strain of E. coli. O157:H7 is a strain of E. coli that evolved by acquiring genes for pathogenic traits. Evidence suggests this occurred by both transduction and the acquisition of a large virulence plasmid via conjugation.​

20

Minutes after mating

Just as in any organism, mutations can arise spontaneously in bacteria. Certain factors, especially those that damage DNA, such as radiation, ultraviolet light, and various chemicals, increase the likelihood of mutation. A typical bacterium such as E. coli contains about 5000 genes. The probability that mutation will occur by chance is about one out of every million copies of a gene. With 5000 genes in a bacterium, approximately 1 out of every 200 bacteria will have a mutation. With adequate food and nutrients, a population of E. coli can double in 20 minutes. Because bacteria multiply so rapidly, mutations can spread rapidly in a population and can change the characteristics of that population in a relatively short time. The ability of prokaryotes to rapidly adapt to selective pressures, such as the presence of an antibiotic in the body, can have adverse effects on human health. A number of antibioticresistant strains of the bacterium Staphylococcus aureus (termed methicillin-resistant Staphylococcus aureus, or MRSA) have been known in hospital settings for some time. Of particular concern among these strains is vancomycin-resistant Staphylococcus aureus (VRSA). This appears to have arisen rapidly by mutation and is alarming because vancomycin is a drug of last resort in treating MRSA, which makes these strains and the infections they cause very difficult to control. Staphylococcus infections provide an excellent example of the way in which mutations in the presence of strong selective pressures can bring about rapid change in bacterial populations.​

Result: The different genes from the donor strain appear in the recipient strain in a linear time sequence. Conclusion: The transfer of genetic information is linear. This sequence can be used to construct a genetic map ordering the genes’ relative positions on the chromosomes. Further Experiments: Can other methods of DNA exchange also be used for genetic mapping?

Figure 27.15 Interrupted mating experiment allows construction of genetic map. An important example in terms of human health involves the Enterobacteriaceae, the family of bacteria to which the common intestinal bacterium E. coli belongs. This family contains

Bacterial growth rates can be exploited to detect carcinogens Due to their rapid growth rates and ease of growth on artificial media in the lab, some bacteria can be used to detect whether a compound is mutagenic. All mutagenic compounds are carcinogenic, and a screening procedure called the Ames test has been widely used to identify mutagenic and, therefore, carcinogenic compounds. Bacteria that can grow on minimal media are called prototrophs. A bacteria that acquires a mutation resulting in an inabiliity to make an essential nutrient, such as an amino acid, is called an auxotroph. Testing for this sort of mutation requires multiple steps, so the insight behind the Ames test is that we can start with chapter

27 Prokaryotes 573

an auxotrophic mutation, and test for reversing this mutation (reversion). This can be done by plating a high density of cells on minimal media where only prototrophs, which have had a reversion, will grow. In practice, a strain of Salmonella typhimurium auxotrophic for the amino acid histidine is treated with a test compound of interest, then plated on minimal medium, which will only support the growth of prototrophs. If the test compound increases the frequency of reversion to prototrophy, it indicates the compound is mutagenic. Of course, no claims can be made about the carcinogenicity of the compound until it is tested in animals at relevant dosages.​

CRISPR provides adaptive defense against viral infection Prokaryotes use a variety of innate defenses against viral infection, including restriction-modification systems (the source of restriction endonucleases) and toxin–antitoxin systems. With the advent of genomics, screens of many prokaryotic genomes revealed a structure of repeated sequences with “spacer regions” that were named CRISPR, for “clustered regularly interspaced short palindromic repeats.” Recently, these have been identified as a form of adaptive protection against viral infection. In response to viral challenge, bacteria and archaea will integrate short segments of viral nucleic acid in these CRISPR loci, then use them to produce an RNA that can be used to guide a complex that degrades viral nucleic acid. Much like restriction endonucleases, the CRISPR system has proved useful for molecular biologists for genome editing (see chapter 17). A 2012 survey found CRISPR loci in 48% of bacterial genomes and 95% of archaeal genomes sequenced. If you consider how viruses outnumber prokaryotic cells in virtually every environmental context, this represents very strong selective pressure for a system that can “remember” previous infection to protect against future infections.

Learning Outcomes Review 27.3

Prokaryotic DNA exchange occurs horizontally, from donor cell to recipient cell. DNA can be exchanged by conjugation, by transduction via viruses, and by transformation through the direct uptake of DNA from the environment. These forms of DNA exchange can be used experimentally to map genes. Variation in prokaryotes also arises by mutation, and the rapid replicative rates of bacteria can be exploited to identify potentially carcinogenic compounds. Overuse of antibiotics has led to selection for resistant organisms. Resistance genes can be transferred, rapidly spreading resistance. Some species of prokaryotes have a form of adaptive protection against future infection by previous agents. ■■

How does the transfer of genetic information in prokaryotes differ from transfer during sexual reproduction?​

574 part V Diversity of Life on Earth

27.4 The

Metabolic Diversity of Prokaryotes

Learning Outcomes 1. Describe the metabolic strategies employed by prokaryotes to obtain carbon, obtain reductants, and transform energy. 2. Compare and contrast the ways prokaryotes can obtain reduced carbon, obtain reductants, and transform energy.

The variation seen in prokaryotes manifests itself most strikingly in biochemical rather than morphological ways. Not only do they have great metabolic diversity, but they also exhibit considerable metabolic flexibility, which is not seen in any other group of organisms.

Prokaryotes have diverse nutritional strategies To meet growth demands, organisms require carbon for building, a source of electrons to use in redox chemistry, and energy for anabolic processes. The nutritional strategy of an organism is simply the ways in which an organism acquires carbon, acquires electrons for redox chemistry, and performs energy transformation. Fortunately, there are only two ways in which carbon, electrons, and energy can each be obtained. Carbon is obtained in reduced forms by heterotrophs, and in oxidized forms (CO2) by autotrophs. Chemotrophs transform energy by oxidizing reduced chemicals obtained from the environment; phototrophs, on the other hand, transform energy by harvesting light. Lithotrophs obtain electrons from reduced inorganic substances in the environment, while organotrophs obtain electrons from reduced carbon sources. Prokaryotes can be classified into five different nutritional types; however, there are times when a particular organism may switch nutritional strategy based on nutritional needs and based on the nutrients available in the environment. This rarely, if ever, happens with eukaryotes. Note that when we name a prokaryote nutritional strategy, we string together into one word the terms that describe the approaches to obtaining carbon, electrons, and energy. The five types are as follows. Chemoorganoheterotrophs. These organisms use reduced (organic) carbon made by producer organisms as sources of carbon, electrons, and energy. They contribute to the biogeochemical cycling of carbon. Nearly all pathogens use this nutritional strategy when they cause disease. The most common sources of reduced carbon in the environments of these organisms are lipids and carbohydrates. All animals are chemoorganoheterotrophs.

Chemolithoheterotrophs. As with chemoorganoheterotrophs, the source of energy and electrons in this nutritional strategy is a reduced chemical in the environment; however, the chemical is inorganic, not organic. The Greek word litho means “stone,” and so these organisms are colloquially known as “stone-eaters” due to the mineral nature of the source of electrons and energy. The source of carbon used by these organisms is a reduced form obtained from the environment. Only prokaryotes utilize this nutritional strategy; they play critical roles in the biogeochemical cycling of many nutrients. Chemolithoautotrophs. The source of carbon in these organisms is the oxidized atmospheric form, CO2. CO2 is reduced to usable organic forms using electrons obtained from reduced inorganic compounds. The oxidation of reduced inorganic compounds also provides the energy required to produce reduced carbon for biosynthesis. Given the use of reduced inorganic nutrients, such as ammonia and sulfur, the use of this nutritional strategy contributes greatly to nutrient cycling in the environment. Photolithoautotrophs. This nutritional strategy also reduces atmospheric CO2 to produce molecules for biosynthesis. The sources of electrons for such reductions are inorganic. You are likely familiar with green plant photosynthesis, which uses water as a source of electrons and produces NADPH to drive the Calvin cycle. Some prokaryotes use water as an electron source; however, others can use hydrogen and different forms of sulfur as electron sources. Prokaryotic photosynthesis can differ significantly from green plant photosynthesis. Photoorganoheterotrophs. This is a particularly unusual nutritional strategy used by prokaryotes such as the purple and green nonsulfur bacteria. The source of energy is light in this strategy; however, organic carbon is used as a source of carbon and of electrons. Regardless of the nutritional strategy used, any energy transformation must ultimately convert one source of chemical potential energy into the form of chemical potential energy usable by all living organisms: ATP.​

Prokaryotes have diverse respirations and fermentations The transformation of energy in one chemical potential energy source into a usable source for the cell involves either photosynthesis, respiration, or fermentation. There can be significant differences in how prokaryotes and eukaryotes perform these processes.​

Differences in respirations between prokaryotes and eukaryotes In eukaryotes, cellular respiration uses an electron transport chain to recycle electron donors into electron acceptors

(primarily NADH and FADH2), reduce oxygen to water, and produce a protonmotive force to drive ATP synthesis (see chapter 7). The final electron acceptor is oxygen, so these respirations are aerobic. Prokaryotic respirations, on the other hand, show a remarkable diversity of both electron donors and electron acceptors. Respirations that use a final electron acceptor other than oxygen are anaerobic respirations. Such acceptors include oxidized substances such as nitrate and sulfate. Some bacteria can even use carbon dioxide as an electron acceptor in a respiration, producing methane gas as a product. Other bacteria are known to reduce organic compounds in their electron transport chains. Prokaryotes also show diversity in the range of electron donors they use in cellular respiration. For example, the chemolithotroph Acidithiobacillius ferrooxidans can oxidize ferrous iron (Fe 2+), elemental sulfur (S 0), and hydrogen sulfide, yielding sulfuric acid. These bacteria can be found in gold mines where ferrous iron is often present and can contribute to the creation of acidic mine runoff that can damage ecosystems.​

Differences in fermentations between prokaryotes and eukaryotes Fermentation may be used in a cell when a terminal electron acceptor is not available to allow respiration. Fermentation can recycle reduced electron acceptors, such as NADH, to their oxidized form, which is needed in oxidation reactions that drive the formation of small amounts of ATP. There is some diversity in the kinds of fermentations seen in eukaryotes: yeasts can ferment sugars to produce ethanol; animal muscle can ferment sugars to produce lactic acid; and under certain conditions, plant cells can ferment sugars to produce ethanol. Prokaryotes, however, show much greater diversity of fermentations. A variety of metabolic pathways used by different prokaryotes can produce different alcohols and acids from the fermentation of pyruvate, the product of glycolysis. Some of these products have industrial or commercial applications and contribute to the flavorings of milk and cheese products. The production of many people’s favorite indulgence, chocolate, is possible only due to the fermentation of cocoa. Perhaps the most unusual fermentation seen in prokaryotes is performed by the Clostridium genus of bacteria. This genus includes species that are industrially valuable due to their production of certain fermentation products, but also includes dangerous human pathogens. C. tetani is the causative agent of tetanus and is introduced into the body most commonly through deep puncture wounds to the skin. The environment deep in a wound is anaerobic, and C. tetani can grow by fermenting the abundant amino acids in tissues. C. botulinum is well known as the pathogenic bacteria producing “Botox” toxin used in cosmetic enhancements to reduce wrinkling of the skin. This bacteria can be introduced into foods during the canning process, and if an abundant source of protein is available in the anaerobic interior of a can, the bacteria can

chapter

27 Prokaryotes 575

ferment the constituent amino acids to support growth. Ingesting the toxin produced by the pathogen along with the food can lead to serious illness and even death. This pathogen is particularly dangerous to young children with poorly developed gastrointestinal (GI) flora.

Learning Outcomes Review 27.4

Prokaryotes exhibit metabolic diversity and flexibility not seen in eukaryotes. Nutritional strategies are named based on understanding the sources of carbon, electrons, and energy used by an organism. There is greater diversity of electron donors and acceptors in prokaryotic respirations than is seen in eukaryotic respirations. Prokaryotic fermentations can metabolize not only pyruvate, but other compounds, to recycle electron donors. The by-products of some fermentations are industrially and commercially valuable. Some pathogens can use specialized fermentations to grow in environments that lead to disease. ■■

How could a bacterial fermentation differ from eukaryotic fermentation in muscle?​

27.5 Microbial

Ecology

Learning Outcomes 1. Describe the role of prokaryotes in biogeochemical nutrient cycling. 2. Explain how animal and plant tissues can be considered microbial ecosystems. 3. Describe the role of prokaryotes in bioremediation.

Prokaryotes were largely responsible for creating the current properties of the atmosphere and the soil through billions of years of their activity. Today, they still affect the Earth and human life in many important ways.​

Prokaryotes play critical roles in nutrient cycling Life on Earth is critically dependent on the cycling of chemical elements between organisms and the physical environments in which they live—that is, between the living and nonliving elements of ecosystems. Nutrient cycling involves two fundamental processes: decomposition, in which nutrients are released to the environment from dead organisms, and fixation, in which oxidized nutrients are made available for use by heterotrophs. Prokaryotes, algae, and fungi play many key roles in this chemical cycling, a process discussed in detail in chapter 56. ​

organisms die and decay, these elements return there. Certain prokaryotes and fungi are decomposers, responsible for the majority of nutrient cycling in ecosystems. Decomposers carry out the decomposition stages of biogeochemical cycles, which release matter from decaying organisms, freeing those nutrients for re-use in the environment. ​

Fixation Other prokaryotes play important roles in fixation, helping to return nutrients from oxidized inorganic forms to reduced organic forms that heterotrophic organisms can use. Carbon. The role of photosynthetic prokaryotes in fixing carbon is critical in biogeochemical cycling of carbon. The organic compounds that plants, algae, and photosynthetic prokaryotes produce from CO2 pass up through food chains to form the bodies of all the ecosystem’s heterotrophs. Some prokaryotes, called methanogens, can also contribute to reduced carbon in the environment by producing methane; methanotrophs, on the other hand, can oxidize methane to CO2. There is a close relationship between the carbon cycle and oxygen production. Ancient cyanobacteria are thought to have added oxygen to the Earth’s atmosphere as a by-product of their photosynthesis to produce reduced carbon compounds. Modern photosynthetic prokaryotes that oxidize water continue to contribute to the production of oxygen and are partly responsible for the reduction of atmospheric and soil CO2 levels. Nitrogen. Less obvious, but no less critical to life, is the role of prokaryotes in cycling nitrogen. The nitrogen in the Earth’s atmosphere is in the form of nitrogen gas. The triple covalent bond that links the two nitrogen atoms is not easy to break. Among the Earth’s organisms, only a very few species of prokaryotes are able to accomplish this, reducing N 2 to ammonia (NH 3), which is used to build amino acids and other nitrogen-containing biological molecules. When the organisms that contain these molecules die, decomposers return nitrogen to the soil as ammonia. This is then converted to nitrate (NO 3−) by nitrifying bacteria, making nitrogen available for plants. The nitrate can also be converted back into molecular nitrogen by denitrifiers that return the nitrogen to the atmosphere, completing the cycle. To fix atmospheric nitrogen, prokaryotes employ an enzyme complex called nitrogenase, encoded by a set of genes called nif (“nitrogen fixation”) genes. The nitrogenase complex is extremely sensitive to oxygen and is found in a wide range of free-living prokaryotes. ​

Decomposition

Animals and plants can be microbial ecosystems

The carbon, nitrogen, phosphorus, sulfur, and other elements of biological systems come from the physical environment, and when

Like all organisms, prokaryotes need places to live that supply their nutritional demands, and where they can evade or deter

576 part V Diversity of Life on Earth

predators. Such homes may be found in the environment, but can also be on or in other organisms. Prokaryotes can exist in a number of types of relationships with organisms in all the kingdoms of life.​

Plants as microbial ecosystems An ecologically important example of a mutually beneficial association between bacteria and plants is the relationship between n­itrogen-fixing bacteria, primarily of the family Rhizobiaceae, and leguminous plants such as peas and soybeans. These bacteria can establish infections in the roots of leguminous plants, where they produce signaling molecules that induce the root to produce a nodule where the bacteria take up residence (figure 27.16). The bacteria are capable of performing nitrogen fixation to produce reduced nitrogen, primarily ammonia, which can be metabolized into organic forms of nitrogen such as amino acids. The bacteria receive photosynthetic metabolites from the plant while providing fixed nitrogen in the form of amino acids. This relationship allows leguminous plants to flourish in soil depleted of reduced nitrogen, and can reduce the amount of fertilizer used agriculturally. Excess fertilizer can lead to an increase in bioavailable nitrogen and phosphorus in agricultural runoff. Not all plant-prokaryote interactions are mutually beneficial, however. Another member of the Rhizobiaceae, Agrobacterium tumefaciens, can establish a parasitic relationship with numerous plant species, including important crop species. Infection by A. tumefaciens causes the formation of a tumorlike mass called a gall. The resulting disease is often called crown gall disease (figure 27.17). Infection is usually initiated at damage on the plant surface; following infection, bacteria use a specialized pilus to transfer a plasmid called the Ti (tumor-inducing) plasmid into plant cells. Once the plasmid is integrated into a plant cell’s genome, a cascade of gene expression from the plasmid stimulates the production of plant hormones that stimulate tissue

Figure 27.16 Nitrogen-fixation nodules on a plant’s root. Worachat Tokaew/Shutterstock

Figure 27.17 Crown gall disease. Nigel Cattlin/Alamy Stock Photo production, resulting in a tumorous gall. The plant cell is also stimulated to produce molecules that other A. tumefaciens recognize and sense as nutrients. Galls damage plant tissues and may result in disrupted water or phloem movement, or promote invasion by other pathogens. Other infections or loss of nutrient transport can lead to death of parts of the plant or the whole plant.​

Animals as microbial ecosystems Due to the ability of some prokaryotes to cause disease, the exploitation of mammals by prokaryotes has been most extensively studied. In addition to the disease-causing relationships we explore in section 27.6, there are many beneficial relationships between mammals and prokaryotes. Prokaryotes and digestion in ruminants. Mammals have evolved diverse structures to facilitate the digestion of diverse foods. Some herbivores have digestive tracts that allow ingested foods to be held long enough that anaerobic bacteria can digest cellulose, the main macromolecule in plant matter. This allows the extraction of essential calories and nutrients from plant material. In ruminants, a specialized digestive organ called the rumen holds cellulose-rich material to be digested by bacteria. The anaerobic rumen bacteria hydrolyze cellulose to glucose, which can be metabolized to make a variety of fatty acids; these can then be absorbed and used as a source of energy and carbon by the animal. Unfortunately for these helpful bacteria, many are digested farther down the GI tract, helping to meet the other nutrient needs of the animal! The human microbiome and its role in health. Humans are excellent hosts for bacteria. Although some of those bacteria may be harmful, the overwhelming majority are beneficial and many play a role in protecting us against pathogens. Humans can host bacteria on their skin and in their respiratory, urogenital, and GI tracts; the collective population of bacteria living on or in a human is called the chapter

27 Prokaryotes 577

human microbiome. Scientists are concerned primarily with the gut microbiome and whether differences between individuals contribute to differences in health or predisposition to certain conditions. Gut microorganisms can play important roles in our ability to digest diets rich in complex polysaccharides. They can also synthesize essential nutrients, such as certain amino acids, that we normally get in our diets. Experiments in mice suggest that there may be a relationship between obesity and gut flora. The precise mechanisms that relate gut microbiota to obesity remain unclear. There seems to be a relationship between the removal of H2 by methanogenic prokaryotes and the stimulation of carbohydrate fermentation by other prokaryotes. This fermentation yields fatty acids that can contribute to an increase in body fat.​

Bioremediation can use prokaryotes The use of organisms to remove pollutants from water, air, and soil is called bioremediation. The normal functioning of sewage treatment plants depends on the activity of microorganisms. In sewage treatment plants, the solid matter from raw sewage is broken down by bacteria and archaea naturally present in the sewage. The end product, methane gas (CH4), is often used as an energy source to heat the treatment plant. Biostimulation—the addition of nutrients such as nitrogen and phosphorus—has been used to encourage the growth of naturally occurring microbes that can degrade crude oil spills. This approach was used to clean up some of the 3 million barrels of oil that spilled into the Gulf of Mexico in 2010 when the Deepwater Horizon oil rig exploded, causing the largest-ever marine oil spill, with a devastating effect on marine life. Similarly, biostimulation has been used to encourage the growth of naturally occurring microbial flora in contaminated groundwater. Current efforts include those concentrated on the use of endogenous microbes such as Geobacter (see figure 27.6) to eliminate radioactive uranium from groundwater contaminated during the Cold War. Chlorinated compounds released into the environment by a variety of sources are another serious pollutant. Some bacteria can actually use these compounds for energy by performing reductive dehalogenation that is linked to electron transport, a process termed halorespiration. Although still in early stages of development, the use of such bacteria to remove halogenated compounds from toxic waste holds great promise.

Learning Outcomes Review 27.5

Prokaryotes are vital to ecosystems for both recycling elements and fixation, or making elements available in organic form. Bacteria are involved in fixation of both carbon and nitrogen and are the only organisms that can fix nitrogen. These nitrogenfixing bacteria may live in symbiotic association with plants. Bacteria are a key component of waste treatment, and they are also being used in bioremediation to remove toxic compounds introduced into the environment. ■■

Does the information about nitrogen fixation shed any light on the practice of crop rotation?​

578 part V Diversity of Life on Earth

27.6 Bacterial

Diseases of Humans

Learning Outcomes 1. Describe how bacterial pathogens cause disease. 2. Explain how Mycobacterium tuberculosis and Helicobacter pylori cause disease. 3. Identify sexually transmitted diseases caused by bacteria.

In the early 20th century, before the discovery and widespread use of antibiotics, infectious diseases killed nearly 20% of all U.S. children before they reached the age of five. Sanitation and antibiotics considerably improved the situation. In recent years, however, we have seen the appearance or reappearance of many bacterial diseases, including cholera, leprosy, tetanus, bacterial pneumonia, whooping cough, diphtheria, and Lyme disease (table 27.1). Members of the genus Streptococcus are associated with scarlet fever, rheumatic fever, pneumonia, necrotizing fasciitis (“flesh-eating disease”), and other infections. Tuberculosis, another bacterial disease, is still a leading cause of death in humans worldwide. We also know now that the bacterium H. pylori can cause both gastric ulcers and cancers associated with the gastric lymphoid tissue. Bacteria have many different methods of spreading through a susceptible population. Tuberculosis and many other bacterial diseases of the respiratory tract are mostly spread through the air in droplets of mucus or saliva. Diseases such as typhoid fever, paratyphoid fever, and bacillary dysentery are spread by fecal contamination of food or water. The pathogens that cause Lyme disease and Rocky Mountain spotted fever are spread to humans by arthropod vectors such as ticks.​

Bacteria cause disease in a variety of ways To cause disease in humans, bacteria must establish an infection, evade the immune system in order to proliferate, and then either cause direct tissue damage, release toxins to cause harm, or evoke an inflammatory response that harms the host. All pathogenic bacteria have specific structures or physiological processes that allow them to use human hosts as environmental niches where they can inadvertently cause disease or even death.​

Colonization The first step in infection is the establishment of the pathogen in the host. The initial site of infection is often where a mucosal surface is exposed to the environment. It is helpful to keep in mind that human urogenital systems are open systems, which allow organisms access to mucosal epithelia that are exposed to the environment. This is also true of human respiratory and gastrointestinal systems. Initially, attachment involves specific and nonspecific adherence of the pathogen to tissue. Attachment is promoted by virulence factors, which are specific genetically determined

TA B L E 2 7.1 Disease

Important Human Bacterial Diseases Pathogen

Vector/Reservoir

Epidemiology

Anthrax

Bacillus anthracis

Animals, including processed hides

Bacterial infection that can be transmitted through contact or ingestion. Rare except in sporadic outbreaks. May be fatal.

Botulism

Clostridium botulinum

Improperly prepared food

Contracted through ingestion or contact with wound. Produces botulinum toxin can be fatal.

Chlamydia

Chlamydia trachomatis

Humans, sexually transmitted disease (STD)

Urogenital infections with possible spread to eyes and respiratory tract. Increasingly common over past 30 years.

Cholera

Vibrio cholerae

Human feces, plankton

Causes severe diarrhea that can lead to death by dehydration; 50% peak mortality rate if untreated. A major killer in times of crowding and poor sanitation; after the 2010 earthquake in Haiti, 180,000 were infected in just 70 days.

Dental caries

Streptococcus mutans, Streptococcus sobrinus

Humans

A dense collection of these bacteria on the surface of teeth leads to secretion of acids that destroy minerals in tooth enamel; sugar alone does not cause caries.

Diphtheria

Corynebacterium diphtheriae

Humans

Acute inflammation and lesions of respiratory mucous membranes. Spread through respiratory droplets. Vaccine available.

Gonorrhea

Neisseria gonorrhoeae

Humans only

STD, on the increase worldwide. Usually not fatal.

Hansen’s disease (leprosy)

Mycobacterium leprae

Humans, armadillos

Chronic infection of the skin; WHO reports worldwide prevalence at 0.2 infections/10,000 individuals, with most occurrences in Southeast Asia. Spread through contact with infected individuals.

Lyme disease

Borrelia burgdorferi

Ticks, deer, small rodents

Spread through bite of infected tick. Lesion followed by malaise, fever, fatigue, pain, stiff neck, and headache. Risk of severe, long-term harm if untreated.

Peptic ulcers

Helicobacter pylori

Humans

Originally thought to be caused by stress or diet, most peptic ulcers now appear to be caused by this bacterium; stomach ulcers can be treated with antibiotics.

Plague

Yersinia pestis

Fleas of wild rodents: rats, squirrels, Killed one-fourth of the population of Europe in the 14th century; endemic in wild prairie dogs. rodent populations of the western United States.

Pneumonia

Streptococcus, Mycoplasma, Chlamydia, Haemophilus

Humans

Acute infection of the lungs; often fatal without treatment. Vaccine for streptococcal pneumonia available.

Tuberculosis

Mycobacterium tuberculosis

Humans

An acute bacterial infection of the lungs, lymph, and meninges. Incidence had been on the rise but is now falling. Infections are complicated by the existence of drug-resistant forms of the bacterium.

Typhoid fever

Salmonella typhi

Humans

A systemic bacterial disease of worldwide incidence. Fewer than 500 cases a year are reported in the United States. Spread through contaminated water or foods (such as improperly washed fruits and vegetables). Vaccines are available for travelers.

Typhus

Rickettsia typhi

Lice, rat fleas, humans

Historically a major killer in times of crowding and poor sanitation; transmitted from human to human through the bite of infected lice and fleas. Peak untreated mortality rate of 70%.

features of a pathogen. These include pili, capsules, S-layers, and cell wall components such as lipopolysaccharide (LPS) and techoic acids. Some pathogens are specific for particular tissues. For example, S. mutans, one of the bacteria involved in plaque formation and dental cavity formation, can attach to the surface of teeth, but not to epithelia. Other pathogens are specific for species. For example, Mycobacterium leprae, the causative agent of leprosy, can grow only in humans, a few species of nonhuman primates, the nine-banded armadillo, and the foot-pad of mice. The specificity of pathogens for tissues and organisms is likely mediated at the

level of colonization, specifically due to the presence of receptors on host tissues to which the pathogen can attach. Immune surveillance mechanisms can rapidly detect the presence of a pathogen in the body and will act to eliminate it unless the pathogen can evade the host immune defenses.​

Immune evasion by pathogens It is not in the interests of a pathogen to kill its host, since the host is the source of nutrition and protection. Therefore, pathogens need strategies to replicate without being eliminated by the host immune system, and without killing the host. Disease and chapter

27 Prokaryotes 579

death are unfortunate side effects of infection, rather than a strategy of most pathogens. To establish relatively long-term infections that allow replication of the pathogen, the host immune system must be evaded. Again, specific virulence factors facilitate this evasion. The first host defense a pathogen must defeat is phagocytosis. Phagocytosis is a complex process involving the attraction of phagocytic cells to the pathogen, engulfment of the pathogen by the phagocyte, and destruction of the pathogen inside the phagocyte (see chapter 50). Different pathogens can evade different parts of this process. Some pathogens take up residence at sites that are inaccessible to phagocytes, such as on the surface of the skin. Other pathogens produce molecules that look like host cell molecules, which they present on their surface. Some pathogens, such as M. tuberculosis, grow so slowly that they fail to elicit an inflammatory response, which would alert phagocytic cells. Other pathogens produce molecules that block the attraction of phagocytes. Perhaps the ultimate form of immune system evasion occurs when pathogens are adapted to exist inside cells, sometimes even immune cells themselves. For example, Lysteria monocytogenes, which can cause listeriosis, can infect and live inside host cells, where it is virtually invisible to the host immune system.

heat and a variety of chemical treatments. These can then be used to induce an immunological response that protects against the effects of infection by pathogens producing the original toxin. Toxoids derived from the diphtheria and tetanus toxins are the basis of several vaccines given that protect against disease caused by infection with Corynebacterium diphtheriae and Clostridium tetani bacteria respectively.​

Invasion

M. tuberculosis is most commonly found in the well-aerated parts of the upper respiratory tract, where it lives inside other cells, most commonly phagocytes. By living inside other cells and inactivating a macrophage’s ability to destroy it, it effectively evades the immune system and persists in the host. It is also possible that due to its very slow growth time, the pathogen rarely alerts the immune system to its presence. Much of the virulence of M. tuberculosis can be attributed to the unusual structure of its cell wall. Although the cell wall of M. tuberculosis contains a small amount of peptidoglycan, the pathogen is classified as neither gram-positive nor gram-negative; rather, the cell wall is characterized by a large amount of unusual lipids called mycolic acids. These lipids contribute to the slow growth of the bacteria and its resistance to many antibiotics, and protect against the damaging effects of many toxic molecules produced inside macrophages. Respiratory symptoms are initially caused by an attempt by the immune system to control the pathogen. Cells recruited to the infection can release toxic substances that cause inflammation and direct damage to respiratory epithelia. If the disease progresses, nodules in the infected tissue called tubercles form, which can block airways in the upper parts of the lungs.

Not all pathogens cause disease at the point of infection; some can produce secreted proteins that break down host tissues to allow the pathogen to spread in the body. These proteins are collectively called invasins and can be a major cause of disease due to tissue damage. Invasins usually cause localized tissue damage to facilitate pathogen spread by degrading molecules such as hyaluronic acid and collagen in connective tissues. Toxins produced by some pathogens may act locally to cause tissue damage; however, some can cause damage distant from the localized site of pathogen growth. This is often the distinction made between invasins and toxins.​

Bacterial toxins Bacterial toxins can be classified as either LPS or protein. LPS, often called endotoxin, is a constituent of the cell wall in gramnegative bacteria. It can be shed into t­issues and bodily fluids during normal bacterial growth, or in larger amounts upon bacterial death and lysis. LPS is pyrogenic, which means that it triggers inflammatory responses resulting in fever. An overwhelming inflammatory response triggered by higher doses of LPS can be lethal. Exotoxins are proteins secreted by the pathogen that cause damage to the host. Some toxins have specific targets, while others have general effects that lead to tissue damage. Tetanus toxin is a specific toxin that attacks a protein involved in the release of a specific neurotransmitter at neuromuscular junctions. This results in a loss of control over muscle contraction, which can produce uncontrollable muscle spasms strong enough to break bone, and ultimately results in paralysis and death. Many exotoxins elicit a strong immune response and can be partly or completely inactivated by host-produced antibodies. Toxoids are exotoxins that have been purified and inactivated by 580 part V Diversity of Life on Earth

Tuberculosis is a leading cause of mortality Tuberculosis (TB) is second only to HIV/AIDS as the number one killer worldwide from a single infectious agent. The World Health Organization (WHO) reports that in 2019, 1.4 million people (including 208,000 infected with HIV) died from TB. Fortunately, infection and mortality rates have been decreasing. The TB bacillus, Mycobacterium tuberculosis, is spread easily in the air in aerosolic droplets produced by the coughing of symptomatic and asymptomatic carriers of the pathogen. Estimates suggest that up to 25% of the world’s population is infected with TB; however, only 5 to 15% of these individuals will ever develop symptoms of disease.​

How M. tuberculosis causes disease

Tuberculosis treatment Most TB patients are placed on multiple, expensive antibiotics for six to twelve months. Alarming outbreaks of multidrug-resistant (MDR) strains of TB have occurred in the United States and worldwide. These MDR strains are resistant to most of the available anti-TB medications. MDR TB is of particular concern because it requires much more time and is more expensive to treat. Also, it is more likely to prove fatal. The basic principles of TB treatment and control are to make sure all patients complete a full course of medication, so that all of the bacteria causing the infection are killed and drug-resistant

strains do not develop. Great efforts are being made to ensure that high-risk individuals who are infected but not yet sick receive preventive therapy under observation. Such programs are approximately 90% effective in reducing the likelihood of developing active TB and spreading it to others. Worldwide, the TB rate is dropping about 2% per year, and between 2015 and 2019 the cumulative reduction was 9%.​

Helicobacter pylori can cause stomach ulcers and cancer For a long time it was believed that stress caused gastric ulcers. This erroneous connection was partly due to the finding that ulcers were common in subjects with high stress, and often in those with unhealthy lifestyles. Robin Warren and Barry Marshall showed that H. pylori not only caused gastric and some duodenal ulcers, but also resulted in gastric cancer and certain types of lymphoma.

Self-inoculation demonstrated the causal link between H. pylori and gastritis In 1979, Warren found that there were large numbers of H. pylori in the stomachs of many patients suffering from gastritis and gastric ulcers. Warren was joined in his work by Marshall and despite their repeatedly culturing the organism from the stomachs of patients suffering from ulcers, their findings were largely dismissed. Because H. pylori infects only primates, they could not use their experimental organism of choice, the mouse, to establish the relationship between the bacterium and disease. It would be unethical to recruit human subjects to be artificially infected with H. pylori, and then measure the development of gastric ulcers. Marshall decided the only way to proceed would be to infect himself. He mixed some H. pylori obtained from the stomach of a patient suffering from gastritis into a broth and drank it. He soon developed gastritis and several other symptoms associated with H. pylori infection of the stomach. In the lab, Marshall biopsied his own stomach and was able to culture H. pylori obtained from his stomach tissue. This was the first of what would be many additional demonstrations that the bacteria was the causative agent for gastritis, and for peptic ulcer disease.

protein, called CagA, interferes with host cell signaling in a way that leads to changes in the structure of the epithelial cells, and ultimately results in cell proliferation and the recruitment of host immune cells. A second protein, called VacA, interferes with cell proliferation and contributes to an inflammatory response. A chronic inflammatory response at the epithelia results in gastritis. A more focused inflammatory response can result in the formation of an ulcer, which in severe cases can become perforated, causing bleeding into the stomach, a serious and life-threatening condition. Short-term treatment of ulcers involves the use of drugs that neutralize or reduce stomach acid production. Gastritis can be reversed by antibiotic treatment that eliminates the pathogen, and damaged tissue will slowly heal. This further supports the causal relationship between the pathogen and the disease.

H. pylori and cancer H. pylori is classified as a type I carcinogen and is responsible for the largest proportion of infectious-agent-caused cancers, which account for approximately 5 to 6% of the global cancer burden. Some individuals with chronic H. pylori infection may develop gastric adenocarcinoma, while others may develop a form of lymphoma. These cancers are likely due to complex interactions between the pathogen, host physiology, and lifestyle factors. CagA and VacA are believed to be important virulence factors in the development of gastric adenocarcinoma due to their interference with normal cell signaling in epithelial cells at the site of colonization.

Many sexually transmitted infections are caused by bacteria A number of bacteria cause sexually transmitted infections (STIs), three particularly important examples of which are gonorrhea, syphilis, and chlamydia (figure 27.18). ​

?

one STI (e.g. chlamydia) to rise as another (e.g., gonorrhea) falls?

550

H. pylori uses multiple virulence factors to cause gastritis

500 450 Number of cases (per 100,000 people)

The stomach is not a favorable environment for a pathogen: stomach acid has a pH of 2 to 3, material is rapidly moved out of the organ, and it has proteases that can damage the organism. H. pylori is able to overcome this hostile environment by swimming through the stomach contents to a layer of mucus that protects the stomach epithelia from its own digestive enzymes and acids. An enzyme produced by the pathogen reduces the viscosity of the mucus layer and allows it to swim into the mucus layer, where it associates with the gastric epithelia. Being present in the mucus lining of the epithelia also prevents the bacteria being passed through the stomach and out of the body in large numbers. In addition, the bacteria produces an enzyme that produces a region of relatively neutral pH around its surface that protects it from the low pH while it migrates to the mucus layer. Once at the epithelia, H. pylori produces a protein that is translocated into the cytoplasm of gastric epithelial cells. This

Inquiry question How is it possible for the incidence of

400 350 300 250

Syphilis Gonorrhea Chlamydia

200 150 100 50 0 1984

1989

1994

1999

2004 Year

2009

2014

2019

Figure 27.18 Trends in sexually transmitted diseases in the United States. chapter

27 Prokaryotes 581

Gonorrhea Gonorrhea is one of the most prevalent communicable diseases in North America. Caused by the bacterium Neisseria gonorrhoeae, gonorrhea can be transmitted through sexual intercourse or any other sexual contact in which body fluids are exchanged, such as oral or anal intercourse. It can also pass from mother to baby during delivery through the birth canal. The incidence of gonorrhea has been on the decline in the United States, although that decline appears to have leveled off. It remains a serious threat worldwide; especially of concern is the appearance of antibiotic-resistant strains of N. gonorrhoeae. ​

Syphilis Syphilis, a very destructive STI, was once prevalent and deadly but is now less common due to the advent of blood-screening procedures and antibiotics. Syphilis is caused by a spirochete bacterium, Treponema pallidum, transmitted during sexual intercourse or through direct contact with an open syphilis sore. The bacterium can also be transmitted from a mother to her fetus, often causing damage to the heart, eyes, and nervous system of the baby. Once inside the body, the disease progresses in four distinct stages. The first, or primary stage, is characterized by the appearance of a small, painless, often unnoticed sore called a chancre. The chancre resembles a blister and occurs at the location where the bacterium entered the body about three weeks following exposure. This sore heals without treatment in approximately four weeks, deceptively indicating a “cure” of the disease, although the bacterium remains in the body. This stage of the disease is highly infectious. The second stage of syphilis, or secondary syphilis, is marked by a rash, a sore throat, and sores in the mouth. The bacteria can be transmitted at this stage through kissing or contact with an open sore. Commonly at this point, the disease enters the third stage, a latent period. This latent stage of syphilis is symptomless and may last for several years. At this point, the person is no longer infectious, but the bacteria are still present in the body, attacking the internal organs. The final stage of syphilis is the most debilitating, as the damage done by the bacteria in the third stage becomes evident. Sufferers at this stage of syphilis experience heart disease, mental deficiency, and nerve damage. ​

Chlamydia Chlamydia is caused by an unusual bacterium. Chlamydia trachomatis is genetically a bacterium but is an obligate intracellular parasite,

much like a virus. It is susceptible to antibiotics but it depends on its host to replicate its genetic material. The bacterium is transmitted through vaginal, anal, or oral intercourse with an infected person. Chlamydia is called the “silent STI” because women usually experience no symptoms until after the infection has become established. In part because of this symptomless nature, the incidence of chlamydia has increased rapidly from 6.5 cases per 100,000 population in 1984 to 539 cases per 100,000 population in 2018. Although 1.76 million cases were reported in 2018, CDC estimates approximately twice as many infections occur as are recorded. The rate is particularly high among young people; the prevalence among 15- to 24-year-olds is three times that among persons 25 to 39 years old. The effects of an established chlamydia infection on the female body are extremely serious. Chlamydia can cause pelvic inflammatory disease (PID), which can lead to sterility and sometimes death. Infection of the male or female reproductive tract by chlamydia can cause heart disease. Chlamydiae produce a peptide similar to one produced by cardiac muscle. As the body’s immune system tries to fight off the infection, it recognizes and reacts to this peptide. The similarity between the bacterial and cardiac peptides confuses the immune system, and T cells attack cardiac muscle fibers, causing inflammation of the heart. The treatment for the disease is antibiotics, usually tetracycline, which can cross the host cell’s plasma membrane to attack intracellular bacteria. Any woman who experiences the symptoms associated with this STI or who is at risk of developing an STI should be tested for the presence of the chlamydia bacterium; otherwise, her reproductive health may be at risk.

Learning Outcomes Review 27.6

Many human diseases are due to bacterial infection, including tuberculosis, streptococcal and staphylococcal infection, and sexually transmitted diseases. Bacterial pathogens produce disease by colonizing a host, causing an inflammatory response or tissue damage, and evading the immune system. Peptic ulcers can be caused by H. pylori, a bacterial inhabitant of the digestive tract. This pathogen can also cause stomach cancer and a type of lymphoma. Bacteria are responsible for many STIs, including gonorrhea, syphilis, and chlamydia. ■■

Why is infection by most pathogens not fatal?

Chapter Review David M. Phillips/Science Source

27.1 Prokaryotic Diversity A brief history of microbiology. Microbiology grew out of the study of infectious disease, and was aided by technology to view the unseen world. Spontaneous generation was disproved by experimentation, and Koch’s postulates provide guidelines to assign a causative agent to a disease. 582 part V Diversity of Life on Earth

Prokaryotes are fundamentally different from eukaryotes. Prokaryotic features include unicellularity, small circular DNA, division by binary fission, lack of membrane-bounded genetic material, single or multiple flagella, and metabolic diversity.

Despite similarities, bacteria and archaea differ. Bacteria and archaea differ in four main ways: plasma membrane structure and chemistry, cell wall architecture, DNA replication, and gene expression. Archaeal lipids have ether instead of ester linkages and can form tetraether monolayers. The cell walls of bacteria contain peptidoglycans, but those of archaea do not. Both bacterial and archaeal DNA have a single replication origin, but the origin and the replication proteins are different. Archaeal initiation of DNA replication and RNA polymerases are more like those of eukaryotes. Classifying prokaryotes Nine clades of prokaryotes have been found so far, but many bacteria have not been studied because they cannot be cultured (figure 27.6).​

27.2 Prokaryotic Cell Structure Prokaryotes are diverse in shape and size. Although there are some unusual exceptions, prokaryotic cells are generally smaller than eukaryotic cells. There are generally three fundamental prokaryotic cell morphologies: rods, cocci, and spirals. Most prokaryotes have cell walls and other external structures. Bacteria are classified as gram-positive or gram-negative based on the Gram stain (figure 27.7). Gram-positive bacteria have a thick peptidoglycan layer in the cell wall that contains teichoic acid (figure 27.8). Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides in their cell wall (figure 27.8). Some bacteria have a gelatinous layer, the capsule, enabling the bacterium to adhere to surfaces and evade an immune response. Other bacteria have S-layers and slime layers. Many bacteria have a slender, rigid, helical flagellum composed of flagellin, which can rotate to drive movement (figure 27.9). Some bacteria have hairlike pili that have roles in adhesion and exchange of genetic information. Some bacteria form highly resistant endospores in response to environmental stress. The internal organization of prokaryotes. In some prokaryotes, invaginated regions of the plasma membrane function in respiration and photosynthesis. The nucleoid region contains a compacted circular DNA with no bounding membrane. Prokaryotic ribosomes are smaller than those of eukaryotes and some antibiotics work by binding to these ribosomes, blocking protein synthesis. Some prokaryotes segregate metabolic functions into regions of the cytoplasm. Those regions might be membrane bound, or surrounded by a protein coat. Other structures are used to concentrate nutrients or provide certain functions such as orienting a bacteria in a magnetic field.​

27.3 Prokaryotic Genetics Transformation is the uptake of DNA from the environment. Transformation occurs when cells take up DNA from the surrounding medium (figure 27.11). It can be induced artificially in the laboratory. Transduction is the transfer of DNA between prokaryotes by viruses. Generalized transduction occurs when viruses package host DNA and transfer it on subsequent infection (figure 27.12). Specialized transduction is limited to lysogenic phage (figure 27.12).

Conjugation depends on the presence of a conjugative plasmid. DNA can be exchanged by conjugation (figure 27.13), which depends on the presence of conjugative plasmids like the F plasmid in E. coli. The F+ donor cell transfers the F plasmid to the F− recipient cell. The F plasmid can also integrate into the bacterial genome. Excision may be imprecise, so that the F plasmid carries genetic information from the host. Antibiotic resistance can be acquired on resistance plasmids. R plasmids have played a significant role in the appearance of strains resistant to antibiotics, such as S. aureus and E. coli O157:H7. Mutation can lead to rapid genetic adaptation. Mutations can occur spontaneously in bacteria due to radiation, UV, and various chemicals. Rapid division rates in many bacteria require a lot of DNA replication, which means that mutations can accumulate rapidly in populations. The rapid genetic change seen in populations of bacteria allows the identification of compounds that elevate the mutation rate and are thus potential carcinogens. CRISPR provides adaptive defense against viral infection. CRISPR systems incorporate DNA from infecting phage and then use this to degrade DNA from future infections.​

27.4 The Metabolic Diversity of Prokaryotes Prokaryotes have diverse nutritional strategies. Nutritional strategies can be classified based on an organism’s carbon, electron, and energy sources. Carbon sources can be reduced carbon from the environment or in the form of atmospheric carbon dioxide; energy sources can be light, reduced organic compounds, or reduced inorganic compounds; and electron sources can be reduced organic or inorganic compounds. Some prokaryotes can use a variety of nutritional strategies, depending on environment conditions. Prokaryotes have diverse respirations and fermentations. Prokaryotes have significantly greater diversity in their ability to perform respirations than do eukaryotes. Eukaryotes are restricted to organic electron donors and oxygen as a terminal electron acceptor. Prokaryotes can use a diversity of reduced electron donors and a variety of oxidized electron acceptors. Eukaryotic fermentation results in acid production, except in fungi and plants, where alcohols can be produced. Prokaryotes can use a much wider range of electron donors and acceptors when performing fermentations to produce acids, alcohols, or a mixture of both.​

27.5 Microbial Ecology Prokaryotes play critical roles in nutrient cycling. Prokaryotes are involved in the recycling of carbon and nitrogen; only bacteria can fix nitrogen. Animals and plants can be microbial ecosystems. Prokaryotes in the guts of ruminants play important roles in liberating carbon and chemical potential energy contained in cellulose. Humans are important hosts for bacteria and many of them provide protection against disease caused by pathogens. The gut microflora of humans is important in nutrient release in ingested foods, and variations in the endogenous flora may impact human health.

chapter

27 Prokaryotes 583

Bioremediation can use prokaryotes. Bacteria can be used to accelerate the breakdown of man-made environmental contaminants.​

M. tuberculosis causes disease, primarily respiratory disease. It triggers a tissue-damaging inflammatory response in respiratory tissue, so that sufferers lose the ability to efficiently exchange oxygen for carbon dioxide and ultimately suffocate.

27.6 Bacterial Diseases of Humans Bacteria cause disease in a variety of ways. To cause disease, pathogenic bacteria must gain entry to the body, colonize at the site of infection, evade the immune system, spread to other sites in the body, and cause damage by the production of toxins or by triggering inflammatory responses. Tuberculosis is a leading cause of mortality. Tuberculosis continues to be a major public health problem. Treatment requires a long course of antibiotics.

Helicobacter pylori can cause stomach ulcers and cancer. Chronic inflammatory responses triggered by H. pylori in the stomach can lead to gastritis, gastric ulcers, and in some individuals, stomach cancer. The pathogen can be eliminated and disease progression reversed by treatment with antibiotics. Many sexually transmitted infections are caused by bacteria. The potentially dangerous sexually transmitted diseases gonorrhea, syphilis, and chlamydia are caused by bacteria.

Visual Summary Prokaryotes

exhibit

some species have

play roles in Beneficial uses to humans

Diversity Ecosystems

for example Cell structure

Genetic information

variation seen in

Transformation Transduction

Cell surface Internal structures

by associating with

variation seen in

Nutrient cycling

Plants and animals

altered in 4 ways

Shape Cell wall

Metabolism

via

Nutritional strategies

Respiration

recycles

Disease

Heterotrophs or autotrophs

Mutation

5 nutritional types

Contamination

can be caused by

Fermentation

Conjugation

Bioremediation to clean up

rely on Microbiomes

and exhibited in

pathogens cause

Carbon fixes

triggers

Nitrogen

Bacterial toxins Inflammatory response

Review Questions David M. Phillips/Science Source

U N D E R S TA N D 1. A main difference between prokaryotes and eukaryotes is that eukaryotic, but not prokaryotic, cells possess a. b. c. d.

membrane-bound genetic material. a cell membrane. cell walls. ribosomes.

584 part V Diversity of Life on Earth

2. A bacteria that obtains carbon from the atmosphere and energy and electrons from reduced inorganic compounds is best characterized as a a. b. c. d.

chemoorganoheterotroph. chemoheterotroph. chemolithoautotroph. photolithoautotroph.

3. Gram-positive and gram-negative bacteria are distinguished based on differences in their a. b. c. d.

cell walls: gram-positives have peptidoglycan, whereas gramnegatives have pseudo-peptidoglycan. plasma membranes: gram-positives have ester-linked lipids, whereas gram-negatives have ether-linked lipids. cell walls: gram-positives have a thick layer of peptidoglycan, whereas gram-negatives have a thin layer. chromosomal structure: gram-positives have circular chromosomes, whereas gram-negatives have linear chromosomes.

4. Bacteria and archaebacteria may be distinguished based on a. b. c. d.

the type of nucleic acid used as genetic material. cell membrane structure and chemistry. the presence and absence of organelles. size.

5. The transfer of very specific genes from one bacterium to another by a virus is called a. b. c. d.

natural transformation. generalized transduction. specialized transduction. conjugation.

6. Tuberculosis is caused by a(n) a. b. c. d.

bacterium. archaebacterium. virus. protist.

7. During respiration, bacteria can use a greater diversity of electron _____ and _____ than can eukaryotics. a. b. c. d.

oxidizers; reducers reducers; oxidizers donors; reducers donors; acceptors​

A P P LY 1. Which of the following structures is unique to prokaryotes? a. b. c. d.

Pili Flagella Cell wall Spores

2. H. pylori, a bacteria found in the stomach, produces an enzyme called urease. Urease converts urea in the stomach to ammonia and carbamate. Carbamate is then converted to carbonic acid and another molecule of ammonia. What is the purpose of the production of urease in helping H. pylori survive stomach acid? a. b. c. d.

Carbonic acid inactivates proteases in the stomach. Ammonia can act as a base to produce bicarbonate that reduces pH. Ammonia can be converted to NH4+ by stomach acid, thus increasing stomach pH. Ammonia can act as an acid to reduce proton concentration in the stomach.

3. Which of the following would reduce the pathogenicity of H. pylori? a. b. c. d.

Mutation that prevents the production of the flagellin protein Mutation that increases the production of urease (see question 2 above) Mutation that increases the production of CagA Mutation that stimulates the production of the enzyme for breaking down mucus

4. Why might the actual incidence of an STI be higher than the reported rates? a. b. c. d.

People with an STI may be too embarrassed to visit their healthcare provider. People with an STI may not have access to health care. Rates of STIs may be high in populations not wanting to report the infection (such as sex workers and drug users). All of the above

5. Pathogens that kill their hosts too quickly lose the opportunity to spread. Which of the following strategies would promote the spread of a pathogen? a. b. c. d.

Irritation of the respiratory epithelia Production of a painless sore on the genitalia that contains the pathogen Production of a painful sore on the genitalia that contains the pathogen a and b

6. Some bacteria are capable of forming NH4+ from nitrogen gas in root nodules of leguminous plants. This is best described as a(n) a. b. c. d.

oxidation. reduction. phosphorylation. carboxylation.

7. The cell membrane of some photosynthetic bacteria is heavily folded to form membrane invaginations. These folds are used to capture light energy and to perform some of the reactions of photosynthesis. Which structure–function relationship in the chloroplast of green plants is analogous to the structure–function relationship of the membrane folds? a. b. c. d.

The stacks of thylakoid membranes, increasing surface area for light capture The increase in volume of the lumen to maximize ATP production by oxidative phosphorylation The reduction in membrane fluidity associated with membrane folding The reduction in heat energy by an increase in surface area of cristae in chloroplasts​

SYNTHESIZE 1. How could an infection with H. pylori be diagnosed given the knowledge that the urease enzyme it produces in the stomach converts urea into ammonia and ultimately into carbon dioxide? 2. Frederick Griffith’s experiments (see chapter 14) played an important role in showing that DNA is the genetic material. Griffith showed that dead virulent bacteria mixed with live nonvirulent bacteria could cause pneumonia in mice. Live virulent bacteria could also be cultured from the infected mice. The difference between the two strains is a polysaccharide capsule found in the virulent strain. Given what you have learned in this chapter, how would you explain these observations? 3. In the 1960s, it was common practice to prescribe multiple antibiotics to fight bacterial infections. It is also often the case that patients do not always take the entire “course” of their antibiotics. Antibiotic resistance genes are often found on conjugative plasmids. How do these factors affect the evolution of antibiotic resistance and of resistance to multiple antibiotics in particular? 4. Soil-based nitrogen-fixing bacteria appear to be highly vulnerable to exposure to UV radiation. Suppose that the ozone level continues to be depleted. What are the long-term effects on the planet? chapter

27 Prokaryotes 585

28 CHAPTER

Protists Chapter Contents 28.1

Eukaryotic Origins and Endosymbiosis

28.2 Overview of Protists 28.3 Characteristics of the Excavata 28.4 Characteristics of the SAR: Stramenopila 28.5 Characteristics of the SAR: Alveolata 28.6 Characteristics of the SAR: Rhizaria 28.7 Characteristics of the Archaeplastida 28.8 Characteristics of the Amoebozoa 28.9 Characteristics of the Opisthokonta

500 μm ©Stephen Durr

Introduction

Visual Outline exhibit

Protists

Significant variation

are Eukaryotes

are organized into Supergroups

for example

differ from prokaryotes by

Cell organization

Eukaryotic cell

Cell structure

Excavata

Cell function

SAR

Prokaryotic cell

Prokaryotic ancestor of eukaryotic cells

having

A nucleus

having

Semiautonomous organelles

Rhizaria

Land plants

Charophytes

Rhodophyta

Foraminifera Cercozoa

Ciliates

Radiolara

Chlorophytes

Alveolata

Dinoflagellates

Apicomplexans

Stramenopila

Brown algae Diatoms Oomycetes

Parabasalid

Diplomonads

Euglenozoa

Archaeplastida

Amoebozoa

Opisthokonta

Amoebozoa

Choanoflagellates

Fungi

SAR

Excavata

Archaea

Animals

Archaeplastida Eubacteria

Amoebozoa Opisthokonta

dividing

By mitosis

For more than half of the history of life on Earth, all life was microscopic. For over 2 billion years, the largest organisms were unicellular prokaryotes. These prokaryotes lacked internal membranes, except for folds in surface membranes of photosynthetic bacteria. The first evidence of a different type of organism is found in tiny fossils in rock 1.5 billion years old. These fossil cells are up to 10 times larger than bacteria and contain internal membranes and what appear to be small, membrane-bounded structures. The complexity and diversity of form among these single cells is astonishing. The step from relatively simple to quite complex cells marks one of the most important events in the evolution of life: the appearance of a new kind of organism, the eukaryote. Protists are a paraphyletic group of eukaryotes that

are not classified as animals, plants, or fungi. Although the group may seem a little strange at first glance, its members, like the unicellular green alga Micrasterias pictured here, play important roles in human health and welfare, ecology, and nutrient cycling in the environment.

28.1 Eukaryotic

Origins and Endosymbiosis

Learning Outcomes 1. List the defining features of eukaryotes. 2. Define endosymbiosis and explain how it relates to the evolution of mitochondria and chloroplasts. 3. Describe how mitosis in fungi and some protists differs from that in other eukaryotes.

Protists were the first eukaryotes. Eukaryotic cells are distinguished from prokaryotes by the presence of a more complex cytoskeleton and compartmentalization. The exact sequence of events that led to large, complex eukaryotic cells is unknown, but several key events are agreed upon. Loss of a rigid cell wall allowed membranes to fold inward, increasing surface area. Membrane flexibility also made it possible for one cell to engulf another.

Fossil evidence dates the origins of eukaryotes In rocks about 1.5 billion years old, scientists have found wellpreserved microfossils that are noticeably different in appearance from earlier, simpler cells. An example of fossil algae is shown in figure 28.1. These cells are much larger than those of prokaryotes and have internal membranes and thicker walls. Early fossils mark a major event in the evolution of life: A new type of organism had appeared. These new cells are called eukaryotes, from the Greek words meaning “true nucleus,” because they possess an internal structure called a nucleus. All organisms are either prokaryotes (which lack a nucleus) or eukaryotes (which possess a nucleus). During discussions of the origin of eukaryotic internal structure, keep in mind that, as discussed in chapter 24, horizontal gene transfer occurred frequently while eukaryotic cells were evolving.

Eukaryotic cells evolved not only through horizontal gene transfer, but also through infolding of membranes and by engulfing other cells. Today’s eukaryotic cell is the result of cutting and pasting of DNA sequences and acquisition of organelles from different species.

The nucleus and endoplasmic reticulum arose from membrane infoldings Many prokaryotes have infoldings of their outer membranes extending into the cytoplasm that serve as passageways to the surface. The network of internal membranes in eukaryotes is called the endoplasmic reticulum (ER), and the nuclear envelope, an extension of the ER network that isolates and protects the nucleus, is thought to have evolved from such infoldings (figure 28.2).

Mitochondria evolved from engulfed aerobic bacteria Bacteria that live within other cells and provide some benefit for their host cells are called endosymbiotic bacteria (endosymbiosis means “living together in close association”). Their widespread presence in nature led biologist Lynn Margulis in the early 1970s to champion the theory of endosymbiosis, which was first proposed by Konstantin Mereschkowsky in 1905. The endosymbiotic theory supports the concept that a critical stage in the evolution of eukaryotic cells involved endosymbiotic relationships with prokaryotic organisms. According to this theory, energy-producing bacteria may have come to reside within larger bacteria or archaebacteria, eventually evolving into what we now know as mitochondria (figure 28.3). One scenario posits that the original host was an anaerobe with hydrogen-dependent metabolic Endoplasmic reticulum (ER) Plasma membrane

Nuclear envelope

Nucleus Infolding of the plasma membrane

Eukaryotic cell Plasma membrane DNA Prokaryotic ancestor of eukaryotic cells Prokaryotic cell

Figure 28.2 Origin of the nucleus and endoplasmic reticulum. Many prokaryotes today have infoldings of the plasma 50 μm

Figure 28.1 Early eukaryotic fossil. Fossil algae that lived in

Siberia 1 bya. Andrew H. Knoll/Harvard University

membrane (see also figure 27.10). The eukaryotic internal membrane system, called the endoplasmic reticulum (ER), and the nuclear envelope may have evolved from such infoldings of the plasma membrane, encasing the DNA of prokaryotic cells that gave rise to eukaryotic cells. chapter

28 Protists 587

Chloroplast

Organelle with four membranes

Eukaryotic cell with chloroplast and mitochondrion

Red algal nucleus lost

Endosymbiosis

Nucleus

Brown alga

Photosynthetic bacterium

Secondary Endosymbiosis

Nucleus Chloroplast with two membranes

Mitochondrion Eukaryotic cell with mitochondrion Aerobic bacterium

Endosymbiosis

Cyanobacteria

Eukaryotic cell Red alga Nucleus Primary Endosymbiosis

Eukaryotic cell

Internal membrane system

Ancestral eukaryotic cell

Figure 28.3 The theory of endosymbiosis. Scientists propose that ancestral eukaryotic cells, which already had an internal system of membranes, engulfed aerobic bacteria, which then became mitochondria within the eukaryotic cell. Chloroplasts also originated this way, with eukaryotic cells engulfing photosynthetic bacteria. pathways, and that the symbiont produced H2 that could be used by the host. Later, the host adapted to an oxygen-rich environment using the symbiont’s respiratory pathways.

Chloroplasts evolved from engulfed photosynthetic bacteria Photosynthetic bacteria were likely engulfed by other, larger bacteria, leading to the evolution of chloroplasts, which are the photosynthetic organelles of plants and algae (figure 28.3). The history of chloroplast evolution illustrates the care that must be taken in phylogenetic studies. All chloroplasts are likely derived from a single line of cyanobacteria, but the organisms that host these chloroplasts are not monophyletic. This apparent paradox is resolved by considering the possibility of secondary, and even tertiary, endosymbiosis. Figure  25.10 explains how red and green algae both obtained their chloroplasts by engulfing photosynthetic 588 part V Diversity of Life on Earth

Figure 28.4 Endosymbiotic origins of chloroplasts in red and brown algae. cyanobacteria. The brown algae most likely obtained their chloroplasts by engulfing one or more red algae, a process called secondary endosymbiosis (figure 28.4). A phylogenetic tree based only on chloroplast gene sequences from brown, red, and green algae reveals a close evolutionary relationship. This tree is misleading, however, because it is not possible to tell just from these data how much the algal lines had diverged at the time they engulfed the same line of cyanobacteria. Nuclear gene sequences, as well as morphological and chemical traits, are more helpful than chloroplast gene sequences in sorting out algal relations.

Endosymbiosis is supported by a range of evidence The fact that we now witness so many symbiotic relationships lends general support to the endosymbiotic theory. Even stronger support comes from the observation that present-day organelles such as mitochondria and chloroplasts contain their own circular DNA, which is remarkably similar to the DNA of bacteria in size and organization. During the billion and a half years in which mitochondria have existed as endosymbionts within eukaryotic cells, most—but not all—of their genes have been transferred to the chromosomes of the host cells. Also, the mRNAs produced from the genes of the mitochondrial genome are translated using ribosomes that are very much like bacterial ribosomes in size and structure. Many antibiotics that inhibit protein synthesis in bacteria also inhibit protein synthesis in mitochondria and

chloroplasts, but not in the cytoplasm. Further supporting the theory of endosymbiosis, chloroplasts and mitochondria replicate by binary fission, just as most bacteria do.

Mitosis evolved in eukaryotes A prokaryote carries its genes on a single circular DNA molecule. In contrast, a eukaryote carries its genes on multiple chromosomes, which are usually present in pairs. During the evolution of eukaryotes, a systematic process needed to be developed to divide the chromosomes and other cell contents during cell division. The separation of chromosomes is called mitosis, whereas the division of the cytoplasm is called cytokinesis. In fungi and in some groups of protists, the nuclear membrane does not dissolve during mitosis, as it does in plants, animals, and most other protists. Consequently, mitosis is confined to the nucleus. When mitosis is complete in these organisms, the nucleus divides into two daughter nuclei, and only then does the rest of the cell divide. In all other eukaryotes, the nuclear membrane breaks down prior to mitosis, so the separation of chromosomes occurs in the cytoplasm rather than in the nucleus. We do not know whether mitosis without nuclear membrane dissolution represents an intermediate step on the evolutionary journey, or simply a different way of solving the same problem. We cannot see the interiors of dividing cells well enough in fossils to be able to trace the history of mitosis.

Learning Outcomes Review 28.1

Eukaryotes are organisms that contain a nucleus and other membrane-bounded organelles. Endoplasmic reticulum and the nuclear membrane are believed to have evolved from infoldings of the outer membranes. According to the endosymbiont theory, mitochondria and chloroplasts evolved from engulfed bacteria that remained intact. Mitochondria and chloroplasts have their own DNA, which is similar to that of prokaryotes. Unlike other eukaryotes, the nuclear membrane does not dissolve during cell division in fungi and some protists. ■■

?

What evidence supports the endosymbiont theory?

Inquiry question How could you distinguish between primary and secondary endosymbiosis by looking at micrographs of cells with chloroplasts?

28.2

Overview of Protists

Learning Outcomes 1. Describe how an organism would be classified as a protist. 2. Recognize the five supergroups that contain the protists. 3. List the two main means of locomotion used by protists.

Unlike other groups of organisms, protists have no unifying features, but rather are characterized by diversity in structure,

genetics, reproductive strategies, and metabolism. Unlike the way many other organisms are classified, a eukaryote is classified as a protist by exclusion. That is, a eukaryote is considered a protist if it lacks the characteristics required to place it with other fungi, plants, or animals. Many protists are unicellular, but numerous colonial and multicellular groups also exist. Most are microscopic, but some are as large as trees. The origin of eukaryotes, which began with ancestral protists, is among the most significant events in the evolution of life.

Eukaryotes are organized into five supergroups that all contain protists Eukaryotes diverged rapidly in a world that was shifting from anaerobic to aerobic conditions. We may never be able to completely sort out the relationships among different lineages during this major evolutionary transition. Applications of a variety of molecular methods are providing insights into the evolutionary relationships among protists. Molecular systematics is helping to sort out the protists, which are now classified in five supergroups: the Excavata; the SAR, comprising the Stramenopila, the Alveolate, and the Rhizaria; the Archaeplastida; the Amoeobozoa; and the Opisthokonta. In efforts to resolve the controversies surrounding protist phylogeny, DNA sequence data are being used to test different hypotheses regarding evolutionary relationships within and between the groups. The phylogenetic tree shown in figure 28.5 represents one of several organizations regarding the relationships between protist lineages, and is but one hypothesis. In this chapter, we explore the diverse world of the protists based on our current understanding of phylogeny (figure  28.5). Understanding the evolution of protists is key to understanding the origins of plants, fungi, and animals. The supergroups encompass all eukaryotic diversity and many protists are closely related to plants, animals, or fungi. The remaining chapters in this diversity unit (see chapters 29 to 34) focus on the monophyletic fungi, plants, and animals that trace back to common ancestors shared with different protist lineages.

Protist cell surfaces vary widely Protists possess a varied array of cell surfaces. Some protists, such as amoebas, are surrounded by only their plasma membrane. All other protists have a plasma membrane with an extracellular matrix (ECM) deposited on the outside of the membrane. Some ECMs form strong cell walls; for instance, diatoms and foraminifera secrete glassy shells of silica.

Protists have several means of locomotion Protists move by diverse mechanisms. Protist movement is chiefly by either flagellar rotation or pseudopods. Many protists wave one or more flagella to propel themselves through the water, and others use banks of short, flagella-like structures called cilia to create water currents for their feeding or propulsion. Pseudopods (Greek, meaning “false feet”) are the chief means of locomotion among amoebas. Other protists have long, thin pseudopods that can be extended or retracted. Because the tips can adhere to adjacent surfaces, the cell can move by a rolling motion, shortening the pseudopods chapter

28 Protists 589

Land plants

Charophytes

Chlorophytes

Rhodophyta

Foraminifera Cercozoa

Radiolara

Ciliates

Dinoflagellates

Rhizaria

Amoebozoa

Opisthokonta

Amoebozoa

Choanoflagellates

Animals

Archaeplastida

Alveolata

Apicomplexans

Stramenopila

Brown algae Parabasalid

Diplomonads

Euglenozoa

Fungi

SAR

Excavata

Archaea

Diatoms Oomycetes

Eubacteria

Figure 28.5 Eukaryotic evolutionary relationships. Analyses of molecular data group the eukaryotes into the five supergroups shown on the top level of the figure. The phylogeny has been simplified to reflect the lack of consensus over deeper branching relationships. Within the eukaryotes, plants, fungi, and animals are monophyletic clades, but protists are paraphyletic. Protist lineages are shaded in gray.

in front and extending those in the rear. Other protists have numerous cilia that act as tiny oars to move the protist through fluid.

Protists have a range of nutritional strategies Protists can be heterotrophic or autotrophic. The heterotrophs obtain energy from organic molecules synthesized by other organisms. Some autotrophic protists are photosynthetic, whereas others are chemoautotrophic. Some heterotrophic protists are phagotrophs; these are organisms that ingest particles of food into vesicles called food vacuoles or phagosomes. Lysosomes fuse with the food vacuoles, introducing enzymes that digest the food particles within. Digested molecules are absorbed across the vacuolar membrane. Another example of the protists’ tremendous nutritional flexibility is seen in mixotrophs, protists that are both phototrophic and heterotrophic.

Protists reproduce asexually and sexually Protists typically reproduce asexually. In addition, some undergo sexual reproduction regularly, whereas others undergo sexual reproduction at times of stress, including during food shortages.

Asexual reproduction Asexual reproduction involves mitosis, but the process often differs from the mitosis in multicellular animals. For example, the nuclear membrane often persists throughout mitosis, with the mitotic spindle forming within the nucleus. 590 part V Diversity of Life on Earth

In some species, a cell simply splits into nearly equal halves after mitosis. Sometimes the daughter cell is considerably smaller than its parent and then grows to adult size—a type of cell division called budding. In schizogony, common among some protists, cell division is preceded by several nuclear divisions. This allows cytokinesis to produce several individuals almost simultaneously. Some protists, even those with delicate cell surfaces, can survive and reproduce asexually in harsh conditions by forming cysts. A cyst is a dormant form of a cell with a resistant outer covering. Inside the cyst, metabolic activity is reduce to virtually zero. However, not all cysts are resistant to all environments. For example, vertebrate parasitic amoebas can form cysts that are quite resistant to gastric acid but will not survive dessication or high temperatures.

Sexual reproduction Most eukaryotic cells also possess the ability to reproduce sexually, something prokaryotes cannot do. Meiosis (see chapter 11) is a major evolutionary innovation that arose in ancestral protists and allows for the production of haploid cells from diploid cells and the generation of genetic diversity. Sexual reproduction is the process of producing offspring by fertilization, the union of two haploid cells, which were generated by meiosis. The great advantage of sexual reproduction is that it allows for frequent genetic recombination, which contributes to the variation required for evolution by natural selection. The evolution of meiosis and sexual reproduction contributed to the tremendous explosion of diversity among the eukaryotes.

Protists are the bridge to multicellularity Diversity in eukaryotes was also promoted by the development of multicellularity. Some single eukaryotic cells began living in association with others, in colonies. Eventually, individual members of the colony began to assume different duties, and the colony began to take on the characteristics of a single individual. Multicellularity has arisen many times among the eukaryotes. Practically every organism big enough to be seen with the unaided eye, including all animals and plants, is multicellular. The great advantage of multicellularity is that it fosters specialization; some cells devote all of their energies to one function in a tissue, other cells to other functions. Few innovations have had as great an influence on the history of life as the specialization made possible by multicellularity.

Learning Outcomes Review 28.2

Eukaryotes fall into five supergroups based on evolutionary relationships: the Excavata; the SAR, comprising the Stramenopila, the Alveolate, and the Rhizaria; the Archaeplastida; the Amoeobozoa; and the Opisthokonta. All protists have plasma membranes, but other cell-surface components, such as deposited extracellular material (ECM), are highly variable. Protists mainly use flagella or pseudopods to propel themselves. Protists may be autotrophs, heterotrophs, or both. Sexual reproduction is common, but asexual reproduction also occurs in many groups. Multicellular organisms likely arose from colonial protists. ■■

Explain how the evolution of meiosis might be linked to the tremendous diversity of protists.

28.3 Characteristics

Excavata

of the

1. List the main features of diplomonads and parabasalids. 2. Give examples of diplomonads and parabasalids. 3. Explain why euglenozoa cannot be classified as either plants or animals. 4. Describe the distinguishing feature of kinetoplastids.

SAR

Amoebozoa

Opisthokonta

Centers for Disease Control and Prevention

3.0 μm

The supergroup Excavata is composed of two monophyletic clades, the diplomonads and the parabasalids, and the euglenozoa. The name Excavata refers to a groove down one side of the cell body in some groups. Parabasalids and diplomonads have multiple flagella; euglenozoans have unique flagella and several have acquired chloroplasts through endosymbiosis. None of the algae are closely related to euglenozoa, a reminder that endosymbiosis is widespread.

Diplomonads lack functional mitochondria The diplomonads, a group of unicellular organisms, are characterized by the absence of functional mitochondria. Other features include the presence of multiple posterior flagella and two haploid nuclei per cell. The parasitic diplomonad, Giardia intestinalis (­figure  28.6), can pass from human to human via contaminated water and causes diarrhea. Mitochondrial genes are found in their nuclei, leading to the hypothesis that Giardia evolved from aerobic ancestors. Electron micrographs of Giardia cells stained with mitochondrial-­specific antibodies reveal degenerate mitochondria that do not participate in cellular respiration.

The distinguishing feature of parabasalids is an undulating membrane that is used for locomotion (figure 28.7). They also use flagella to propel themselves. Unlike diplomonads, parabasalids have semifunctional mitochondria and have one nucleus per cell. The parabasalid group includes an ecologically diverse array of species. Some live in the gut of termites and digest cellulose, the main component of the termite’s wood-based diet. The symbiotic relationship is one layer more complex because these parabasalids have a symbiotic relationship with bacteria that also aid in the digestion of cellulose. The persistent activity of these three symbiotic organisms from three different kingdoms can lead to the collapse of a home built of wood or recycle tons of fallen trees in a forest. Another parabasalid,

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Foraminifera

Ciliates

Archaeplastida Rhizaria

Radiolara

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Parabasalids

Diplomonads

Stramenopila

diplomonad lacks a functional mitochondrion. Dr. Stan Erlandsen/

Parabasalids have undulating membranes

Learning Outcomes

Excavata

Figure 28.6 Giardia intestinalis. This parasitic

Figure 28.7  Undulating membrane characteristic of parabasalids. Trichomoniasis

is caused by this parasite species, Trichomonas vaginalis. David M. Phillips/Science Source

0.8 μm chapter

28 Protists 591

Trichomonas vaginalis, causes trichomoniasis, a common sexually transmitted disease in humans.

Euglenozoans change shape while swimming The most distinguishing feature of euglenozoans is apparent when they swim. Their bodies change shape, alternating between being stretched out and being rounded up. Euglenozoans can change shape because they lack a cell wall. Instead, strips of protein encircle the cell making up the pellicle. These strips can slide over each other, pro­viding flexibility. Euglenozoa diverged early and were among the earliest free-living eukaryotes to possess mitochondria. Both free-living euglenids and parasitic kinetoplastids are considered euglenozoans.

Euglenids Euglenids clearly illustrate the impossibility of distinguishing plantlike protists from animal-like protists. About one-third of the approximately 170 genera of euglenids have chloroplasts and are fully autotrophic; the others lack chloroplasts, ingest their food, and are heterotrophic. Some euglenids with chloroplasts may become heterotrophic in the dark; the chloroplasts become small and nonfunctional. If put back in the light, they may become green within a few hours. Photosynthetic euglenids may sometimes feed on dissolved or particulate food. Reproduction in the euglenids is asexual and occurs by mitotic cell division. The nuclear envelope remains intact throughout mitosis. No sexual reproduction is known to occur in this group. In Euglena (figure 28.8), the genus for which the euglenid phylum is named, two flagella are attached at the base of a flask-shaped

Figure 28.8  Euglenids. 

a. Micrograph of Euglena gracilis. b. Diagram of Euglena. Paramylon granules are areas where food reserves are stored. Andrew Syred/ Science Source

a.

6 μm Stigma

Second flagellum

Reservoir

Contractile vacuole Basal bodies

Paramylon granule Nucleus

Chloroplast

b. 592 part V Diversity of Life on Earth

Mitochondrion Pellicle

Flagellum

opening called the reservoir, which is located at the anterior end of the cell. One of the flagella is long and has a row of very fine, short, hairlike projections along one side. A second, shorter flagellum is located within the reservoir but does not emerge from it. Contractile vacuoles collect excess water from all parts of the organism and empty it into the reservoir, which helps regulate osmotic pressure within the organism. A light-sensitive stigma, also seen in the green algae (Chlorophyta), helps photosynthetic euglenids move toward light. Cells of Euglena contain numerous small chloroplasts. These chloroplasts, like those of the green algae and plants, contain chlorophylls a and b, together with carotenoids. Although the chloroplasts of euglenids differ somewhat in structure from those of green algae, they probably had a common origin. Euglena’s photosynthetic pigments are light-­sensitive (figure 28.9). It seems likely that euglenid chloroplasts evolved from a symbiotic relationship through ingestion of green algae.

Parasitic kinetoplastids A second major group within the euglenozoa is the kinetoplastids. The name kinetoplastid refers to a unique, single mitochondrion in each cell. Each mitochondrion has minicircles and maxicircles of DNA that are linked together into complex chains. (Remember that prokaryotes have circular DNA, and mitochondria had prokaryotic origins.) Some genes found in the kinetoplastid genome are responsible for an unusual form of RNA processing where uridine is inserted into or deleted from transcripts to correct frameshifts that would otherwise interfere with expression of the RNA. Parasitism has evolved multiple times within the kinetoplastids. Trypanosomes are a group of kinetoplastids that cause many serious human diseases, the most familiar being trypanosomiasis, also known as African sleeping sickness, which causes extreme lethargy and fatigue (figure 28.10). Leishmaniasis, which is transmitted by sand flies infected with the protist parasite Leishmania, is a trypanosomic disease that causes skin sores and in some cases can affect internal organs, leading to death. About 1.3 million new cases are reported each year. The rise in leishmaniasis in South America correlates with the move of infected individuals from rural to urban environments, where there is a greater chance of spreading the parasite. Chagas disease is caused by Trypanosoma cruzi. At least 90 million people, from the southern United States to Argentina, are at risk of contracting T. cruzi. The parasite is carried by an insect vector, the triatomine bug. The bug is found in dwellings constructed from materials such as mud, adobe, and straw. The insect bites the host and while feeding on its blood, deposits feces. The parasite can then enter the body of the host from the insect’s feces when the host scratches and breaks the skin, or transfers the parasite to a mucous membrane. The disease can also be passed from mother to child during childbirth, during blood transfusions, and from organ transplants using infected organs. Chagas disease can lead to severe cardiac and digestive problems in humans and domestic animals, but it appears to be tolerated in wild mammals. Control is especially difficult because of the unique att­ri­butes of these parasites. For example, tsetse fly–transmitted trypanosomes have evolved an elaborate genetic mechanism for repeatedly changing the antigenic nature of their protective glycoprotein coat, thus dodging the antibodies their hosts produce against them (see chapter 51). Only a single gene out of some

SCIENTIFIC THINKING Hypothesis: Euglena cells do not retain photosynthetic pigments in a dark environment. Prediction: If light-grown Euglena are transferred to the dark, photosynthetic pigments will be degraded and new pigment will not be made. Test: Grow Euglena under normal light conditions. Transfer the culture to two flasks. Take a sample from each flask and measure the amount of photosynthetic pigment in each. Maintain one flask in the light and transfer the other to the dark. After several days, extract the photosynthetic pigments from each flask, and compare amounts with each other and with initial levels.

Partition into two flasks. Grow culture of Euglena under light.

Allow growth for several days. Take a sample from each flask and quantify photosynthetic pigment.

Put one flask in dark, and expose the other to light.

Quantify photosynthetic pigment in each flask.

Result: Photosynthetic pigment levels are lower in the dark-grown flask than in the light-grown one. Pigment levels in the dark-grown flask are lower than at the beginning of the experiment. Pigment levels in the light-grown flask are unchanged. Conclusion: The hypothesis is supported. Maintenance of Euglena in the dark resulted in a loss of photosynthetic pigment. Pigments were degraded in the dark-grown flask. Further Experiments: Transfer dark-grown flasks back to the light and measure changes in pigment levels over time. Are original pigment levels restored after growth in light?

Figure 28.9 Effect of light on Euglena photosynthetic pigments. Figure 28.10 A kinetoplastid. 

a. Trypanosoma among red blood cells. The nuclei (dark-staining bodies), anterior flagella, and undulating, changeable shape of the trypanosomes are visible in this photomicrograph. b. The tsetse fly, shown here sucking blood from a human arm, can carry trypanosomes. (a) Dr. Myron G. Schultz/Centers for

Trypanosome Blood cell

Disease Control and Prevention; (b) Edward S. Ross

a.

20 μm

b.

1000 variable-surface glycoprotein (VSG) genes is expressed at a time. A VSG gene is usually duplicated and moved to 1 of about 20 expression sites near the end of the chromosome where it is transcribed. In the guts of the flies that spread them, trypanosomes are noninfective. When they are ready to transfer to the skin or bloodstream of their host, trypanosomes migrate to the salivary glands and acquire the thick coat of glycoprotein antigens that protect them from the host’s antibodies. Later, when they are taken up by a tsetse fly, the trypanosomes again shed their coats. The production of vaccines against such a system is complex. Releasing sterilized flies to impede the reproduction of populations is another technique being tried to control the fly population. Traps made of dark cloth and scented like cows, but poisoned with insecticides, have likewise proved effective. The sequencing of the genomes of the three kinetoplastids described earlier revealed a core of common genes in all three. The devastating toll all three take on human life could be alleviated by the development of a single drug targeted at one or more of the core proteins shared by the three parasites.

Learning Outcomes Review 28.3

Excavata are grouped based on cytoskeletal structure and DNA sequence similarities. Within the excavates, diplomonads lack functional mitochondria but may contain mitochondrial genes. They are unicellular, have two nuclei, and move with flagella; an example is Giardia. Parabasalids produce semifunctional mitochondria and use flagella and undulating membranes for locomotion; an example is Trichomonas. The euglenids include phototrophs and heterotrophs. Some members have chloroplasts that remain nonfunctional unless light is present, and some phototrophs may feed if food particles are present. The kinetoplastids contain a single mitochondrion with two types of DNA and the ability to edit RNA. Trypanosomes are diseasecausing kinetoplastids. ■■ ■■

In what type of habitat would it be useful to use undulating membranes for locomotion? How does a contractile vacuole regulate osmotic pressure in a Euglena cell? chapter

28 Protists 593

28.4 Characteristics

Stramenopila

single-celled photosynthetic red alga. The Stramenopila, a monophyletic clade within the SAR supergroup, is composed of the brown algae, the diatoms, and the ooymycetes (“water molds”).

of the SAR:

Brown algae include large seaweeds

Learning Outcome 1. Distinguish members of Stramenopila from one another.

SAR

Amoebozoa

Opisthokonta

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Rhizaria

Foraminifera

Ciliates

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Parabasalids

Diplomonads

Stramenopila

Archaeplastida

Radiolara

Excavata

Brown algae are the most conspicuous seaweeds in many northern regions of the world (figure 28.12). The haplodiplontic life cycle of the brown algae is marked by alternation of generations between a multicellular sporophyte (diploid) and a multicellular gametophyte (haploid) (figure 28.13). Some sporophyte cells go through meiosis and produce spores. These spores germinate and undergo mitosis to produce the large individuals we recognize, such as the kelps. The gametophytes are often much smaller, filamentous individuals, perhaps a few centimeters in width. Even in an aquatic environment, transport can be a challenge for the very large brown algal species. Distinctive transport cells that stack one upon the other enhance transport within some species (see figure 23.11). However, even though the large kelp look

The SAR supergroup includes the monophyletic clades Stramenopila, Alveolata, and Rhizaria. The Stramenopila and Alveolata share a more recent common ancestor than does either with the Rhizaria. For this reason, the Stramenopila and Alveolata are often discussed together. But for clarity, our discussion will tackle each of the groups separately, starting with the Stramenopila. The name Stramenopila refers to unique, fine hairs found on the flagella of members of this group (figure 28.11), although some species have lost the hairs during evolution. The members of the group are largely photosynthetic and are thought to have originated over a billion years ago when an ancestor (which was also the common ancestor of the related Alveolata) engulfed a

18,500×

Figure 28.11 Stramenopiles have very fine hairs on their flagella. David Patterson for Maple Ferryman Pty Ltd. 594 part V Diversity of Life on Earth

Figure 28.12 Brown alga. The giant kelp, Macrocystis pyrifera, grows in relatively shallow water along the coasts throughout the world and provides food and shelter for many different kinds of organisms. Ethan Daniels/Shutterstock

Zygote (2n)

FERT

Sperm

MI

TO

SIS

TION ILIZA

Egg

Developing sporophyte

n

Gametophytes (n)

2n

Germinating zoospores Zoospores (n) MEIO SIS

Sporophyte (2n)

Figure 28.13 Haplodiplontic cycle of Laminaria, a brown alga. Multicellular haploid and diploid stages are found in this life cycle,

although the male and female gametophytes are quite small.

like plants, it is important to realize that they do not contain the complex tissues such as xylem that are found in plants.

Diatoms are unicellular organisms with double shells Diatoms, members of the phylum Chrysophyta, are photosynthetic, unicellular organisms with unique double shells made of opaline silica, which are often strikingly marked (­figure 28.14). The shells

of diatoms are like small boxes with lids, one half of the shell fitting inside the other. Their chloroplasts, containing chlorophylls a and c, as well as carotenoids, resemble those of the brown algae and dinoflagellates. Diatoms produce a unique carbohydrate called chrysolaminarin. Some diatoms move by using two long grooves, called raphes, which are lined with vibrating fibrils (figure  28.15). The exact mechanism is still being unraveled and may involve the ejection of mucopolysaccharide streams from the raphe that propel the diatom.

Raphe

90 μm

Figure 28.14 Diatoms. These different radially symmetrical

diatoms have unique silica, two-part shells. Dennis Kunkel Microscopy/ Science Source

5 μm

Figure 28.15 Diatom raphes are lined with fibrils that aid in locomotion. Andrew Syred/Science Source chapter

28 Protists 595

Learning Outcomes Review 28.4

Most members of the stramenopiles have fine hairs on their flagella. Brown algae are large seaweeds that provide food and habitat for marine organisms. They undergo an alternation of generations. Diatoms are unicellular with silica in their cell wall, which forms a shell with two halves. Some can propel themselves. Oomycetes are unique in the production of zoospores that bear two unequal flagella. ■■

How could you distinguish between the sporophyte and the gametophyte of a brown alga?

28.5 Characteristics

Alveolata

SAR

Amoebozoa

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

The second clade within the SAR that we look at contains the alveolates, which include the dinoflagellates, apicomplexans, and ciliates, all of which have a common lineage but diverse modes of locomotion. A common trait is the presence of flattened vesicles called alveoli (hence the name Alveolata) stacked in a continuous layer below their plasma membranes (figure 28.16). The alveoli may function in membrane transport, similar to the way Golgi bodies function.

Dinoflagellates are photosynthesizers with distinctive features Most dinoflagellates are photosynthetic unicells with two flagella. Dinoflagellates live in both marine and freshwater environments. Some dinoflagellates are luminous and contribute to the twinkling or flashing effects seen in the sea at night, especially in the tropics, while other species contribute to harmful algal blooms in marine environments that can lead to human disease. The flagella, protective coats, and biochemistry of dinoflagellates are distinctive, and the dinoflagellates do not appear to be directly related to any other phylum. Plates made of a cellulose-like material, often encrusted with silica, encase the

of the SAR: Alveolar sac

Learning Outcomes 1. Identify the distinguishing feature of the members of alveolates. 2. Explain the function of the apical complex in apicomplexans. 3. Describe the characteristics of the ciliates.

596 part V Diversity of Life on Earth

Opisthokonta

Rhizaria

Foraminifera

Ciliates

Archaeplastida

Radiolara

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Stramenopila

Parabasalids

All oomycetes are either parasites or saprophytic (organisms that live by feeding on dead organic matter). At one time, these organisms were considered fungi, which is the origin of the term water mold and why their name contains the root word -mycetes. Oomycetes are distinguished from other protists by the structure of their motile spores, or zoospores, which bear two unequal flagella, one pointed forward and the other backward. Zoospores are produced asexually in a sporangium. Sexual reproduction involves the formation of male and female reproductive organs that produce gametes. Most oomycetes are found in water, but their terrestrial relatives are plant pathogens. Phytophthora infestans, which causes late blight of potatoes, was one of multiple factors, including weather and oppressive social conditions, that contributed to the Irish potato famine of 1845–1849. During the famine, about 400,000 people starved to death or died of diseases complicated by starvation, and about 2 million Irish immigrated to the United States and elsewhere. Another oomycete, Saprolegnia, is a fish pathogen that can cause serious losses in fish hatcheries. When these fish are released into lakes, the pathogen can infect amphibians and kill millions of amphibian eggs at a time at certain locations. This pathogen is thought to contribute to the phenomenon of amphibian decline.

Excavata

Diplomonads

Oomycetes, the “water molds,” have some pathogenic members

Apical complex 1 μm

Figure 28.16 An alveolus is a continuum of vesicles just below the plasma membrane of dinoflagellates, apicomplexans, and ciliates. The apical complex of the alveolus forces the parasite into host cells. Vern Carruthers, David Elliott

Ptychodiscus Noctiluca Gonyaulax Ceratium

Figure 28.17 Some dinoflagellates. Noctiluca, which lacks the heavy cellulose armor characteristic of most dinoflagellates, is one of the bioluminescent organisms that cause the waves to sparkle in warm seas. In the other three genera, the shorter, encircling flagellum is seen in its groove, with the longer one projecting away from the body of the dinoflagellate. (Not drawn to scale.)

dinoflagellate cells (figure  28.17). Grooves at the junctures of these plates usually house the flagella, one encircling the cell like a belt, and the other perpendicular to it. By beating in their grooves, these flagella cause the dinoflagellate to spin as it moves. Most dinoflagellates have chlorophylls a and c, in addition to carotenoids, so that in the biochemistry of their chloroplasts, they resemble the diatoms and the brown algae. Most dinoflagellates, like brown algae and diatoms, have chloroplasts containing chlorophylls a and c in addition to carotenoids. Possibly this lineage acquired such chloroplasts by forming endosymbiotic relationships with members of those groups. Although sexual reproduction does occur under starvation conditions, dinoflagellates reproduce primarily by asexual cell division. Asexual cell division relies on a unique form of mitosis in which the permanently condensed chromosomes divide within a permanent nuclear envelope. After the numerous chromosomes duplicate, the nucleus divides into two daughter nuclei. Also, the dinoflagellate chromosome is unique among eukaryotes in that the DNA is not generally complexed with histone proteins. In all other eukaryotes, the chromosomal DNA is complexed with histones to form nucleosomes, structures that represent the first order of DNA packaging in the nucleus (see chapter 10). How dinoflagellates maintain distinct chromosomes with a small amount of histones remains a mystery.

Overgrowth of dinoflagellates The poisonous and destructive “red tides” that occur frequently in coastal areas are often associated with great population explosions, or “blooms,” of dinoflagellates, whose pigments color the water (figure 28.18). The blooms are most often triggered by excess nutrients from agricultural and other human activity. Red tides have a profound, detrimental effect on the fishing industry worldwide. Some 20 species of dinoflagellates produce powerful neurotoxins that inhibit the diaphragm, causing respiratory failure in many vertebrates. When the toxic dinoflagellates are abundant, many fishes, birds, and marine mammals may die. Humans consuming affected fish or shellfish will also be ingesting neurotoxins that accumulate in the tissues of these animals. One type of poisoning, paralytic shellfish poisoning, can be caused by dinoflagellate blooms on both the east and the west coasts of the United States.

Figure 28.18 Red tide. Although small in size, huge populations of dinoflagellates can color the sea and release toxins into the water. In this case, the dinoflagellates are green. Visual&Written SL/Alamy Stock Photo

Apicomplexans include the malaria parasite Apicomplexans are spore-forming parasites of animals. They are called apicomplexans because of a unique arrangement of fibrils, microtubules, vacuoles, and other cell organelles at one end of the cell, termed an apical complex (see ­figure 28.6). The apical complex is a cytoskeletal and secretory complex that enables the apicomplexan to invade its host. The best-known apicomplexan is the malarial parasite Plasmodium. (The use of the genome sequence of the parasite and the mosquito that carries it is discussed in chapter 24.)

Plasmodium and malaria Malaria, a disease with 218 million cases and 405,000 deaths in 2018, is caused by the Plasmodium parasite. The parasite is transmitted via bites of infected Anopheles mosquitoes. Like other apicomplexans, Plasmodium has a complex life cycle involving sexual and asexual phases and alternation between different hosts, in this case mosquitoes (Anopheles gambiae) and humans (­figure 28.19). Efforts to eradicate malaria have focused on (1) eliminating the mosquito vectors; (2) developing drugs to poison the parasites that have entered the human body; and (3) developing vaccines. From the 1940s to the 1960s, wide-scale applications of dichlorodiphenyltrichloroethane (DDT) killed mosquitoes in the United States, Italy, Greece, and certain areas of Latin America. For a time, the worldwide elimination of malaria appeared possible. But this hope was soon crushed by the appearance of DDT-resistant mosquitoes in many regions. Furthermore, the use of DDT has had serious environmental consequences. In addition to the problems with resistant strains of mosquitoes, strains of Plasmodium have appeared that are resistant to the drugs historically used to kill them, including quinine. An experimental vaccine called RTS,S has been developed to prevent Plasmodium infection of humans. The vaccine targets a protein called CSP, and is thought to work by preventing the sporozoites from infecting hepatocytes shortly after transmission by a mosquito. Blocking this stage of the infection would prevent the parasite from making its way from the liver to the blood to establish a systemic infection. Although RTS,S is only 30% effective in chapter

28 Protists 597

Inside Mosquito

Inside Mammal

1. While feeding, mosquito injects Plasmodium sporozoites into human.

6. The gametocytes develop into gametes and reproduce sexually, forming sporozoites within the mosquito.

2. Sporozoites enter the liver, reproduce asexually and release merozoites into the bloodstream.

Sporozoite

Host’s liver cell

Sporozoite Oocyst

Fertilization

Merozoite

Gametes

Host’s red blood cell Gametocyte

5. Gametocytes are ingested by another, previously uninfected mosquito.

4. Certain merozoites develop into gametocytes.

3. Merozoites multiply inside red blood cells and are released. The cycle repeats.

Figure 28.19 The life cycle of Plasmodium. Plasmodium, the apicomplexan that causes malaria, has a complex life cycle that alternates between mosquitoes and mammals.

children and infants, pilot evaluations in Ghana, Kenya, and Malawi, involving hundreds of thousands of infants, began in 2018.

Gregarines Gregarines are another group of apicomplexans that use their distinctive apical complex to attach themselves in the intestinal epithelium of arthropods, annelids, and mollusks. Most of the gregarine body, aside from the apical complex, is in the intestinal cavity, and nutrients appear to be obtained through the apicomplexan attachment to the cell (figure 28.20).

Toxoplasma Using its apical complex, Toxoplasma gondii invades the epithelial cells of the human gut. Most individuals infected with the parasite mount an immune response, preventing any permanent damage. In the absence of a fully functional immune system, however, Toxoplasma can damage brain (figure  28.21), heart, and skeletal tissues, in addition to gut and lymph tissue, during extended infections. Individuals with AIDS are particularly susceptible to Toxoplasma infection. If a pregnant women touches a litter box of an infected cat, Toxoplasma parasites from the cat can, if ingested, cross the placental barrier and harm the developing fetus.

Ciliates are characterized by their mode of locomotion As the name indicates, most ciliates feature large numbers of cilia (tiny beating hairs). Their cilia are usually arranged either in 598 part V Diversity of Life on Earth

400×

Figure 28.20 Gregarine entering a cell. De Agostini Picture Library/Getty Images

Figure 28.21  Micrograph of a cyst filled with Toxoplasma.  Toxoplasma can enter the brain and form cysts filled with slowly replicating parasites. Prof. David J.P.

Ferguson, Oxford University

10 μm

longitudinal rows or in spirals around the cell. Cilia are anchored to microtubules beneath the plasma membrane (see chapter 5), and beat in a coordinated fashion. In some groups, the cilia have specialized functions, becoming fused into sheets, spikes, and rods that may then function as mouths, paddles, teeth, or feet. The ciliates have a pellicle, a tough but flexible outer covering, that enables them to squeeze through or move around obstacles.

Anterior contractile vacuole Macronucleus Micronucleus Cytoproct Posterior contractile vacuole

Food vacuole

Micronucleus and macronucleus All known ciliates have two different types of nuclei within their cells: a small micronucleus and a larger macronucleus (­figure  28.22). DNA in the macronucleus is transcribed for the daily activities of the organism. The macronucleus is typically polyploid and may contain up to 1000 copies of each chromosome. The micronucleus is diploid and used only as the germ line for sexual reproduction. DNA in the micronucleus is not transcribed.

Gullet Cilia Pellicle

Figure 28.22 Paramecium. The main features of this ciliate include cilia, two nuclei, and numerous specialized organelles.

Vacuoles Ciliates form vacuoles for ingesting food and regulating water balance. Food first enters the gullet, which in Paramecium is lined with cilia (figure 28.22). Food is pulled into the cell by the beating action of the cilia, passing into food vacuoles, where enzymes and hydrochloric acid aid in its digestion. Afterward, the vacuoles empty their waste contents through special pores in the pellicle called cytoprocts, which are essentially exocytotic vesicles that appear periodically when solid particles are ready to be expelled.

7. One of these micronuclei is the precursor of the micronucleus for that cell, and the other eventually gives rise to the macronucleus.

The contractile vacuoles, which regulate water balance, periodically expand and contract as they empty their contents to the outside of the organism.

Conjugation: Exchange of micronuclei Like most ciliates, Paramecium undergoes a sexual process called conjugation, in which two cells remain attached to each other for up to several hours (figure 28.23).

1. Two Paramecium individuals of different mating types come into contact.

Micronucleus Macronucleus

O

SI

S

350× M

EI

2. The diploid micronucleus in each divides by meiosis to produce four haploid micronuclei.

Haploid micronucleus 2n SIS MITO

5. In each individual, the new micronucleus fuses with the micronucleus already present, forming a diploid micronucleus.

n

Diploid micronucleus

CONJUGATION

6. The macronucleus disintegrates, and the diploid micronucleus divides by mitosis to produce two identical diploid micronuclei within each individual.

MI

TO

SIS

4. Mates exchange micronuclei.

3. Three of the haploid micronuclei degenerate. The remaining micronucleus in each divides by mitosis.

Figure 28.23 Life cycle of Paramecium. In sexual reproduction, two mature cells fuse in a process called conjugation. M. I. Walker/

Science Source

chapter

28 Protists 599

“Killer” strains Paramecium strains that kill other, sensitive strains of Paramecium long puzzled researchers. Initially, killer strains were believed to have genes coding for a substance toxic to sensitive strains. The true source of the toxin turned out to be an endosymbiotic bacterium in the “killer” strains. If this bacterium is engulfed by a “nonkiller” strain, the toxin is released, and the sensitive Paramecium dies.

Learning Outcomes Review 28.5

All members of the Alveolata contain flattened vesicles called alveoli. Dinoflagellates have pairs of flagella arranged perpendicularly to each other, which causes them to swim with a spinning motion. Blooms of dinoflagellates result in red tides. Apicomplexans are animal parasites that produce a structure called an apical complex, which is composed of cytoskeleton and secretory structures and aids in penetrating their host. The ciliates are unicellular, heterotrophic protists with cilia used for feeding and propulsion. ■■

SAR

Amoebozoa

Opisthokonta

Rhizaria

Actinopoda have silica exoskeletons Many Rhizaria have amorphous shapes, with protruding pseudopods that are constantly changing form. Roughly characterized as amoeboid, these same pseudopod-shaped bodies are also found in other protist supergroups. One group of Rhizaria, however, has more distinct structures. Members of the phylum Actinopoda, often called radiolarians, secrete glassy exoskeletons made of silica. These skeletons give the unicellular organisms a distinct shape, exhibiting either bilateral or radial symmetry. The shells of different species form many elaborate and beautiful shapes, with pseudopods extruding outward along spiky projections of the skeleton (fig­ure 28.24), which are supported by microtubule-based structures.

of the SAR:

Learning Outcome 1. Distinguish between the shells of most foraminifera and those of diatoms.

600 part V Diversity of Life on Earth

Animals

Rhizaria has been combined with Stramenopila and Alveolata to form the supergroup SAR, which is currently thought to be monophyletic. The Rhizaria include two monophyletic groups—radiolarians and forams—and a third has been proposed, the cercozoans. These three groups are morphologically quite different from one another, and until recently they had not been grouped together or linked to a particular branch of the protist phylogenic tree. As with much of our rapidly changing picture of protist phylogeny, this grouping will undoubtedly be refined as future analyses come into sharper focus.

What would be a major difficulty in finding a poison to fight the malaria-causing protist Plasmodium?

28.6 Characteristics

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Rhizaria

Foraminifera

Ciliates

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Parabasalids

Stramenopila

Archaeplastida

Radiolara

Excavata

Diplomonads

Paramecia have multiple mating types. Only cells of two different genetically determined mating types can conjugate. Meiosis in the micronuclei produces several haploid micronuclei, and the two partners exchange a pair of their micronuclei through a cytoplasmic bridge that forms between the conjugating cells. In each conjugating individual, the new micronucleus fuses with one of the micronuclei already present in that individual, resulting in the production of a new diploid micronucleus. After conjugation, the macronucleus in each cell disintegrates, and the new diploid micronucleus undergoes mitosis, thus giving rise to two new identical diploid micronuclei in each individual. One of these micronuclei becomes the precursor of the future micronuclei of that cell, while the other micronucleus undergoes multiple rounds of DNA replication, becoming the new macronucleus. This complete segregation of the genetic material is unique to the ciliates and makes them ideal organisms for the study of certain aspects of genetics.

10

Figure 28.24 Actinosphaerium with needle-like pseudopods. Wim Van Egmond/SPL/Science Source

Foraminifera fossils created huge limestone deposits Members of the phylum Foraminifera are heterotrophic marine protists. They range in size from microscopic to several centimeters. They resemble tiny snails and can form 3-m-deep layers in marine sediments. Characteristic of the group are pore-studded shells (called tests) composed of organic materials usually reinforced with grains of calcium carbonate, sand, or even plates from shells of echinoderms or spicules (minute needles of calcium carbonate) from sponge skeletons. Depending on the building materials they use, foraminifera may have shells of very different appearance. Some of them are red, salmon, or yellow-brown. Most foraminifera live in sand or are attached to other organisms, but two families consist of free-floating planktonic organisms. Their tests may be single-chambered, but are more often multichambered, and they sometimes have a spiral shape resembling that of a tiny snail. Thin cytoplasmic projections called podia emerge through openings in the tests (­figure 28.25). Podia are used for swimming, gathering materials for the tests, and feeding. Foraminifera eat a wide variety of small organisms. The life cycles of foraminifera are extremely complex, alternating between haploid and diploid generations. Foraminifera have contributed massive accumulations of their tests to the fossil record for more than 200 million years. Because of the excellent preservation of their tests and the striking differences among them, forams are very important as geological markers. The pattern of occurrence of different forams is often used as a guide in searching for oil-bearing strata. Limestones all over the world, including the famous White Cliffs of Dover on the south coast of Great Britain, are rich in forams (figure 28.26).

Cercozoa locomote with flagella or pseudopods Cercozoa are a morphologically diverse group of primarily soil protists. Some rely on flagella for locomotion, but others extend

Figure 28.26 White Cliffs of Dover. The limestone that forms these cliffs is composed almost entirely of fossil shells of protists, including foraminifera. Markus Keller/age fotostock

pseudopods for amoeboid movement. Some have silica-based shells made up of scales or plates. A cercozoan, Paulinella chromatophora, may have ingested a cyanobacterium as recently as 60 mya. Except for plants, this is the only known case of primary endosymbiosis. Consequently, this species is being investigated for clues to the evolution of endosymbiosis.

Learning Outcomes Review 28.6

Rhizaria move with the aid of needle-like pseudopods. Many members of the foraminifera occupy marine habitats. Most of them have calcium carbonate shells and are responsible for large fossil deposits of limestone, whereas cercozoans and radiolarians have silica shells. ■■

How would you determine if amoeboid locomotion using pseudopods was a good trait to use in reconstructing protist phylogenies?

28.7 Characteristics

Archaeplastida

8 μm

Figure 28.25 A representative of the foraminifera. 

Podia, thin cytoplasmic projections, extend through pores in the calcareous test, or shell, of this living foram. Peter Parks/Oceans-Image/

of the

Learning Outcomes 1. List the major characteristics of red algae. 2. Describe how humans use red algae. 3. Explain why charophytes are considered the closest relatives of land plants.

Photoshot/Newscom

chapter

28 Protists 601

SAR

Archaeplastida

Amoebozoa

Opisthokonta

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Rhizaria

Foraminifera

Ciliates

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Parabasalids

Diplomonads

Stramenopila

Radiolara

Excavata

0.1 mm

Archaeplastids acquired their chloroplasts through primary endosymbiosis, unlike the brown algae, in which secondary endosymbiosis resulted in photosynthetic cells (see figures 28.3 and 28.4). As noted in section 28.1, the common origin of the chloroplast genome in all the algae complicates phylogenetic research, especially because some of the chloroplast genes moved into the nuclear genome. Extensive comparisons of brown, red, and green algal DNA sequences are consistent with a closer evolutionary relationship between the red and the green algae than between either of those groups and the brown algae, which belong to the chromalveolates. A smaller group of algae, the glaucophytes, are also grouped with the Archaeplastida. Glaucophyte plastids were acquired through primary endosymbiosis and have a peptidoglycan (sugar and amino acid polymer) layer that harks back to the cyanobacterial endosymbiont. The green algae have two groups—Chlorophyta and Charophyta—with the Charophyta sharing a common ancestor with the land plants. Land plants are the focus of chapters 29 and 30, with other Archaeplastida discussed here.

Data analysis To construct a phylogeny with brown, red, and green algae, look for the amount of sequence variation in several genes from the chloroplast genomes of species in each of the groups and nuclear genomes. Few nucleotide differences are to be found among the chloroplast genes. More nucleotide differences occur between the brown algal and either the red or the green algal nuclear genes than between the red and green nuclear genes. Draw a phylogeny and explain your reasoning.

Rhodophyta are photosynthetic, multicellular, marine algae The red algae, formally known as Rhodophyta, include more than 6000 species, ranging in size from microscopic organisms to Schizymenia borealis with blades as long as 2 m (­figure 28.27). Many of the algae that build coral reefs are in this group. Sushi rolls are wrapped in nori, a red alga. Agarose, isolated from the cell wall of certain species of red algae, can be used to make agar, a common thickening agent in some foods. Agar is also used to produce solid growth media for culturing microorganisms. 602 part V Diversity of Life on Earth

Figure 28.27 Red algae come in many forms and sizes. (Top left) Steven P. Lynch; (Top Right) Steven P. Lynch; (Bottom) Premaphotos/ Alamy Stock Photo

This lineage lacks flagella and centrioles and has the accessory photosynthetic pigments phycoerythrin, phycocyanin, and allophycocyanin, which are often red, and mask the green color of chlorophyll. These pigments allow the algae to absorb blue and green light, which penetrate relatively deeply into ocean waters. Consequently, red algae can live at great depths in marine environments. As with the brown algae, red algae have both haploid and diploid phases in their life cycles.

Chlorophytes and charophytes are green algae Green algae have two distinct lineages: the chlorophytes, discussed here, and another lineage, the charophytes, that gave rise to the land plants (see figure  28.5). The chlorophytes are of special interest here because of their unusual diversity and lines of specialization. The chlorophytes have an extensive fossil record dating back 900 million years. Modern chlorophytes closely resemble land plants, especially in the biochemical composition of their chloroplasts. They contain chlorophylls a and b, as well as carotenoids.

Unicellular chlorophytes Early green algae probably resembled Chlamydomonas reinhardtii, diverging from land plants over 1 bya (figure 28.28). Individuals are microscopic, green, and rounded, and they have two flagella at the anterior end. They are soil dwellers that move rapidly in water by beating their flagella in opposite directions. Most individuals of Chlamydomonas are haploid. Chlamydomonas reproduces

Reproductive cells – Gamete Asexual reproduction – Strain

Vegetative cells

+ Gamete

MITOSIS MITOSIS

n + Strain

M

EI

OS

2n

Pairing of positive and negative mating strains

FERTILIZATION

20 nm

Figure 28.29 Volvox. This chlorophyte forms a colony in

IS

which some cells are specialized for reproduction. Stephen Durr

Zygospore (diploid)

cells, each cell having two flagella. Only a small number of the cells are reproductive. Some reproductive cells may divide asexually, bulge inward, and give rise to new colonies that initially remain within the parent colony. Other reproductive cells produce gametes for sexual reproduction.

Haplodiplontic life cycles in multicellular chlorophytes

4 μm

Figure 28.28 Chlamydomonas life cycle. This single-celled chlorophyte has both asexual and sexual reproduction. Unlike multicellular green plants, gamete fusion is not followed by mitosis. Aaron J. Bell/Science Source asexually as well as sexually, but because it is always unicellular, the life cycle is not haplodiplontic (figure 28.28). Several lines of evolutionary specialization have been derived from organisms such as Chlamydomonas, including the evolution of nonmotile, unicellular green algae. Chlamydomonas is capable of retracting its flagella and settling down as an immobile unicellular organism if the pond in which it lives dries out. Some common algae found in soil and bark, such as Chlorella, are essentially like Chlamydomonas in this trait, but they do not have the ability to form flagella. The genome of Chlamydomonas reinhardtii has been seq­­uenced, and this organism has become a workhorse for comparative genetic studies and gene function analyses. In fact, it is a widely used host for recombinant protein expression and the biomanufacture of antibodies, immunotoxins, hormones, and industrial enzymes.

Cell specialization in colonial chlorophytes Multicellularity arose many times in the eukaryotes. Colonial chlorophytes provide examples of cellular specialization, an aspect of multicellularity. The most elaborate of these organisms is Volvox (figure 28.29), a hollow sphere made up of a single layer of 500 to 60,000 individual

Haplodiplontic life cycles are found in some chlorophytes and the streptophytes, which include both charophytes and land plants. Ulva, a multicellular chlorophyte, has identical gametophyte and sporophyte generations that consist of flattened sheets two cells thick (figure 28.30). Unlike the charophytes, none of the ancestral chlorophytes gave rise to land plants.

?

Inquiry question Are Ulva gametes formed by meiosis? Explain your response.

Charophytes: Closest Relatives to Land Plants Charophytes are also green algae, and they are distinguished from chlorophytes by their close phylogenetic relationship to the land plants. Currently, the molecular evidence from rRNA and DNA sequences favors the charophytes as the green algal clade most closely related to land plants. Charophytes also have haplontic life cycles. Identifying which of the charophyte clades is sister (most closely related) to the land plants puzzled biologists for a long time, as the charophyte algae fossil record is scarce. The two candidate charophyte clades have been the Charales and the Coleochaetales (figure 28.31). Both lineages are primarily freshwater algae, but the Charales are huge, relative to the microscopic Coleochaetales. Both clades have similarities to land plants. Coleochaete and its relatives have cytoplasmic linkages between cells called plasmodesmata, which are found in land plants. The species Chara in the Charales undergoes mitosis and cytokinesis like land plant cells. Sexual reproduction in both relies on a large, nonmotile egg and flagellated sperm. These gametes are more similar to those of land plants than to many charophyte relatives. Both charophyte clades form green mats chapter

28 Protists 603

around the edges of freshwater ponds and marshes. One species must have successfully inched its way onto land through adaptations to drying.

−Gametangia −Gametophyte (n) −

Learning Outcomes Review 28.7

Gametes

Red algae vary greatly in size and produce accessory pigments that may give them a red color. They lack centrioles and flagella, and they reproduce using an alternation of generations. Humans use red algae as a food and to prepare a thickening agent called agar. The chlorophytes have chloroplasts very similar to those of land plants. Specializations in this group include nonmotile, unicellular species that can tolerate drying and colonial organisms that exhibit some cell specialization. Charophytes are the green algae that are most likely sisters to the land plants based on molecular evidence, morphology, and reproduction.

+ +Gametangia FE

+Gametophyte (n) n

RT

Z ILI

I AT

ON

Zygote 2n

Germinating zygote

Spores

■■

M

EI

OS

IS

■■

Why would you expect to get different results from phylogenetic analysis of nuclear DNA versus plastid DNA? What major barrier must be overcome for sexual reproduction of land-based organisms?

Sporophyte (2n) Sporangia

28.8 Characteristics

Amoebozoa

of the

Learning Outcomes 1. Explain how amoebas move. 2. Describe characteristics of plasmodium. 3. Distinguish between cellular and plasmodial slime molds.

0.1 mm

604 part V Diversity of Life on Earth

Opisthokonta

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Foraminifera

Ciliates

Amoebozoa

Rhizaria

Radiolara

Dinoflagellates

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Alveolata

Archaeplastida

50 μm

Figure 28.31 Chara, a member of the Charales, and Coleochaete, a member of the Coleochaetales, represent the two clades most closely related to land plants. (left) blickwinkel/Alamy Stock Photo; (right) Lee W. Wilcox

Brown algae

Coleochaete

SAR Stramenopila

Parabasalids

Chara

Excavata

Diplomonads

Figure 28.30 Life cycle of Ulva. This chlorophyte alga has a haplodiplontic life cycle. The gametophyte and sporophyte are multicellular and identical in appearance. (bottom) Dr. Diane S. Littler

Amoebas move from place to place by means of their pseudopods. As mentioned in section 28.7, pseudopods are flowing projections of cytoplasm that extend and pull the amoeba forward or engulf food particles. An amoeba puts a pseudopod forward and then

Figure 28.32 

Amoeba proteus. 

The projections are pseudopods; an amoeba moves by flowing cytoplasm into them. Michael Abbey/Science Source

60 μm

flows into it (­figure 28.32). Microfilaments of actin and myosin similar to those found in muscles are associated with these movements. The pseudopods can form at any point on the cell body so that it can move in any direction. The amoebas in the Amoebozoa supergroup are most closely related to the Opisthokonta, which include the close relatives of fungi and animals. Members of these two supergroups have a single flagellum, in contrast with two or more in the other supergroups. Amoebozoans and Opisthokonts are collectively referred to as Unikonts, with “uni” referring to the single flagellum; however, they are distinct supergroups.

Most amoeba are free-living, but some are parasitic Found in the soil, as well as in freshwater, most of these Amoebozoans are free-living and important to the soil ecosystem. A few rare cases of a species acting as a human pathogen have been reported. In immune-suppressed individuals, the Acanth­amoeba enters the body through a wound and is able to cross the blood– brain barrier into the brain. Once in the brain, inflammation and death follow.

Plasmodial slime molds are multinucleate A plasmodial slime mold streams along as a plasmodium, a nonwalled, multinucleate mass of cytoplasm that resembles a moving mass of slime (figure 28.33). This form is called the feeding phase, and the plasmodium may be orange, yellow, or another color.

Figure 28.34 Sporangia of a plasmodial slime mold. 

These Arcyria sporangia are found in the phylum Myxomycota. Eye of Science/Science Source

Plasmodia show a back-and-forth streaming of cytoplasm that is very conspicuous, especially under a microscope. They are able to pass through the mesh in cloth or simply to flow around or through other obstacles. As they move, they engulf and digest bacteria, yeasts, and other small particles of organic matter. A multinucleated Plasmodium cell undergoes mitosis synchronously, with the nuclear envelope breaking down, but only at late anaphase or telophase. Centrioles are absent. When either food or moisture is in short supply, the plasmodium migrates relatively rapidly to a new area. Here it stops moving and either forms a mass in which spores differentiate or divides into a large number of small mounds, each of which produces a single, mature sporangium, the structure in which spores are produced. These sporangia are often beautiful and extremely complex in form (figure 28.34). The spores are highly resistant to unfavorable environmental influences and may last for years if kept dry.

Cellular slime molds exhibit cell differentiation The cellular slime molds have become an important group for the study of cell differentiation because of their relatively simple developmental systems (figure  28.35). The individual organisms behave as separate amoebas, moving through the soil and ingesting bacteria. When food becomes scarce, the individuals aggregate to form a moving “slug.” Cyclic adenosine monophosphate (cAMP) is sent out in pulses by some of the cells, and other cells move in the direction of the cAMP to form the slug. In the cellular slime mold Dictyostelium discoideum, this slug goes through morphogenesis to make stalk and spore cells. The spores then go on to form a new amoeba if they land in a moist habitat.

Learning Outcomes Review 28.8

At least three lineages of slime mold exist. Cellular slime molds are multicellular, and plasmodial slime molds consist of large multinucleate single cells.

Figure 28.33 A plasmodial protist. This multinucleate pretzel slime mold, Hemitrichia serpula, moves about in search of the bacteria and other organic particles that it ingests. Eye of Science/Science Source

■■

Would you say that cellular slime molds are closely related to plasmodial slime molds?

chapter

28 Protists 605

Spores are released

Figure 28.35 Development in Dictyostelium discoideum, a cellular slime mold. (1) First, a

1 Spores germinate into feeding amoebas

6 Amoebas encyst as spores in the fruiting body

Spores

Lack of food triggers amoebas to aggregate

Amoebas

Amoebas aggregate

Fruiting body 2 Amoebas form a mound Cells differentiate into a stalk

5 Slug stops and culmination begins

Slug migrates toward light

28.9 Characteristics

Opisthokonta

4 Mound forms a 2–3 mm long slug

animals. Choanoflagellates have a single emergent flagellum surrounded by a funnel-shaped, contractile collar composed of closely placed filaments, a structure that is exactly matched in the sponges, which are animals. These protists feed on bacteria strained out of the water by their collar. Colonial forms resemble freshwater sponges (figure 28.36). The close relationship of choanoflagellates to animals was further demonstrated by the strong homology between a surface receptor (a tyrosine kinase receptor) found in choanoflagellates and sponges. This surface receptor initiates a signaling pathway involving phosphorylation (see chapter 9).

of the

Learning Outcome 1. Describe the evolutionary significance of the choanoflagellates.

SAR

Archaeplastida

Amoebozoa

Opisthokonta

Animals

Choanoflagellida

Fungi

Amoebozoa

Land plants

Charophytes

Chlorophytes

Cercozoa

Rhodophyta

Rhizaria

Foraminifera

Ciliates

Dinoflagellates

Alveolata

Apicomplexans

Diatoms

Oomycetes

Euglenozoa

Brown algae

Parabasalids

Diplomonads

Stramenopila

Radiolara

Excavata

3 Mounded amoebas produce a tip-like structure

spore germinates, forming an amoeba that feeds and reproduces until the food runs out. At that point, amoebas aggregate and move toward a fixed center. (2) The aggregated amoebas begin to form a mound. (3) The mound produces a tip and begins to fall sideways. (4) Next, the aggregate forms a multicellular “slug,” 2–3 mm long, that migrates toward light. (5) The slug stops moving, and a process called culmination begins. Cells differentiate into stalk and spore cells. (6) In the mature fruiting body, amoebas become encysted as spores.

Learning Outcomes Review 28.9

The choanoflagellates are believed to be the closest relatives of animals. Colonial forms are similar to freshwater sponges, and both organisms have a homologous cell-surface receptor. ■■

What other types of studies might connect choanoflagellates with sponges?

Figure 28.36 

Colonial choanoflagellates resemble their close animal relatives, the sponges. David Fungi and animals are more closely related to each other than to plants because they share a common ancestor, which leads them to be grouped as Opisthokonts. Of particular interest in the Opisthokonts are the choanoflagellates, unicellular ­organisms that are most like the common ancestor of the sponges and, indeed, all 606 part V Diversity of Life on Earth

Patterson for Maple Ferryman Pty Ltd.

30 μm

Chapter Review ©Stephen Durr

28.1 Eukaryotic Origins and Endosymbiosis Fossil evidence dates the origins of eukaryotes. Although eukaryotes may have arisen earlier, the fossil evidence of their appearance dates back to 1.5 bya.

Euglenozoans change shape while swimming. Free-living euglenids, exemplified by Euglena, can produce chloroplasts to carry out photosynthesis in the light. They contain a pellicle and move via anterior flagella. Kinetoplastids are parasitic and are distinctive in having a single, unique mitochondrion with two types of circular DNA.

The nucleus and endoplasmic reticulum arose from membrane infoldings (figure 28.2).

28.4 Characteristics of the SAR: Stramenopila

Mitochondria evolved from engulfed aerobic bacteria. According to the theory of endosymbiosis, ancestral eukaryotic cells engulfed aerobic bacteria, which then became mitochondria (figure 28.3).

Brown algae include large seaweeds. Brown algae are typically large seaweeds that have a haplodiplontic life cycle, producing gametophyte and sporophyte stages (figure 28.13).

Chloroplasts evolved from engulfed photosynthetic bacteria. Chloroplasts are believed to have arisen when ancestral eukaryotic cells engulfed photosynthetic bacteria (figure 28.4). Brown algae arose through a secondary endosymbiotic event when a red alga was engulfed by a single-celled organism. Endosymbiosis is supported by a range of evidence. In support of endosymbiosis, several organelles are found to contain their own DNA, which closely resembles that of prokaryotes. Over a billion and a half years, many of the chloroplast and mitochondrial genes have migrated to the nuclear genome. Mitosis evolved in eukaryotes. Mechanisms of mitosis vary among organisms, suggesting that the process did not evolve all at once.

28.2 Overview of Protists Eukaryotes are organized into five supergroups that all contain protists. Molecular systematics is helping to sort out the protists, which are now grouped into five supergroups: the Excavata; the SAR, comprising the Stramenopila, the Alveolata, and the Rhizaria; the Archaeplastida; the Amoeobozoa; and the Opisthokonta (figure 28.5). Monophyletic clades have been identified among the protists. Protist cell surfaces vary widely. Extracellular material may cover the plasma membrane. Protists have several means of locomotion. Protists mainly use flagella or pseudopods for locomotion, although many other means of propulsion are found. Protists have a range of nutritional strategies. Protists include phototrophs, heterotrophs (phagotrophs or osmotrophs), and mixotrophs capable of both modes. Protists reproduce asexually and sexually. Protists can reproduce asexually by mitosis, budding, or schizogony. They may also carry out sexual reproduction. Protists are the bridge to multicellularity. Colonial protists may be the precursors of multicellular organisms.

28.3 Characteristics of the Excavata Diplomonads lack functional mitochondria. Diplomonads are unicellular, move with flagella, and have two nuclei. Parabasalids have undulating membranes. Parabasalids use flagella and undulating membranes for locomotion.

Diatoms are unicellular organisms with double shells. Diatoms have silica in their cell walls. Each diatom produces two overlapping glassy shells that fit like a box and lid. Oomycetes, the “water molds,” have some pathogenic members. Oomycetes are parasitic and are unique in the production of asexual spores (zoospores) that bear two unequal flagella.

28.5 Characteristics of the SAR: Alveolata Dinoflagellates are photosynthesizers with distinctive features. Dinoflagellates have pairs of flagella arranged so that they swim with a spinning motion. Blooms of dinoflagellates cause red tides (figure 28.18). Apicomplexans include the malaria parasite. Apicomplexans are spore-forming animal parasites (figure 28.19). They have a unique arrangement of organelles at one end of the cell, called the apical complex, which is used to invade the host. Ciliates are characterized by their mode of locomotion. Ciliates are unicellular, heterotrophic protists that use numerous cilia for feeding and propulsion. Each cell has a macronucleus and a micronucleus. Micronuclei are exchanged during conjugation (figure 28.23).

28.6 Characteristics of the SAR: Rhizaria Actinopoda have silica exoskeletons. Glassy exoskeletons made of silica give radiolarians a distinct shape. Microtubules support pseudopods that extrude toward the tips of the spiky exoskeleton. Foraminifera fossils created huge limestone deposits. The Foraminifera are heterotrophic marine protists with pore-studded shells primarily formed by deposits of calcium carbonate. Cercozoa locomote with flagella or pseudopods. Like the marine radiolarians, cercozoans have silica exoskeletons, but most are found in soils. Cercozoans ingest green algae and may provide clues to the evolution of endosymbiosis.

28.7 Characteristics of the Archaeplastida Rhodophyta are photosynthetic, multicellular, marine algae. Red algae produce accessory pigments that may give them a red color. They lack centrioles and flagella, and they reproduce using an alternation of generations. Chlorophytes and charophytes are green algae. Unicellular chlorophytes include Chlamydomonas, which has two flagella, and Chlorella, which has no flagella and reproduces asexually. Volvox is an example of a colonial green alga; some cells are specialized for producing gametes or for asexual reproduction. It may represent a chapter

28 Protists 607

step on the way to multicellularity. Multicellular chlorophytes can have haplodiplontic life cycles. Ulva has sporophyte and gametophyte generations; however, the chlorophytes did not give rise to land plants although they are the closest relatives to land plants. Both groups within the charophytes, Charales and Coleochaetales, have plasmodesmata, cytoplasmic links between cells. They also undergo mitosis and cytokinesis like terrestrial plants. Charophytes are most closely related to the land plants of all the algae.

28.8 Characteristics of the Amoebozoa Most amoebas are free-living, but some are parasitic. Many protists are free-living and found in the soil and freshwater ecosystems, but a few species are pathogenic to humans.

Plasmodial slime molds are multinucleate. Plasmodia in their feeding phase are macroscopically visible masses of oozing slime (figure 28.33). These colorful large cells undergo repeated rounds of mitosis without cell division. Cellular slime molds exhibit cell differentiation. Cellular slime molds such as Dictyostelium discoideum can signal and interact with neighboring cells, resulting in differentiated cell types in an aggregate organism.

28.9 Characteristics of the Opisthokonta Colonial choanoflagellates are structurally similar to freshwater sponges, and molecular similarities have been found.

Visual Summary Significant variation

Protists

exhibit

are

Eukaryotes

are organized into

for example

differ from prokaryotes in

Supergroups are

Cellular organization Size

Excavata

SAR

Archaeplastida

Cell surface Locomotion Nutritional strategies

Diplomonads Parabasalids Euglenozoans

Rhodophyta Chlorophytes Charophytes

gave rise to

Amoebozoa

Opisthokonta

having

having

Mitosis

Mitochondria and chloroplasts

Nucleus and endoplasmic reticulum

Animals

possibly arising from

possibly arising from

Endosymbiosis

Infoldings of cell membrane

gave rise to

Free-living, parasitic slime molds

includes

Fungi

Plants

Reproduction

dividing by

Choanoflagellates Stramenopila

Rhizaria Alveolata

Review Questions ©Stephen Durr

U N D E R S TA N D 1. Fossil evidence of eukaryotes dates back to a. 2.5 bya. b. 1.5 bya.

c. 2.5 mya. d. 1.5 mya.

2. The products of budding are a. b.

two cells of equal size. two cells, one of which is smaller than the other.

608 part V Diversity of Life on Earth

c. d.

many cells of equal size. many cells of variable size.

3. Both diplomonads and parabasalids a. b. c. d.

contain chloroplasts. have multinucleate cells. lack mitochondria. have silica in their cell walls.

4. Trypanosomes are examples of a. euglenids. b. diplomonads.

c. parabasalids. d. kinetoplastids.

5. The function of the apical complex in apicomplexans is to a. b. c. d.

propel the cell through water. penetrate host tissue. absorb food. detect light.

6. If a cell contains a pellicle, it a. b. c. d.

can change shape readily. is shaped like a sphere. is shaped like a torpedo. must have a contractile vacuole.

7. Stramenopila are a. tiny flagella. b. large cilia.

c. small hairs on flagella. d. pairs of large flagella.

8. Choose all of the following that exhibit an alternation of multicellular generations. a. Dinoflagellates b. Brown algae

c. Red algae d. Diatoms

9. Choose all of the following that are photosynthetic. a. Diatoms b. Ciliates

c. Apicomplexans d. Dinoflagellates

10. Which is most likely the ancestor of animals? a. Trypanosomes b. Diplomonads

c. Ciliates d. Choanoflagellates

11. When food is scarce, cells of this organism communicate with each other to form a multicellular slug. a. Cellular slime molds b. True amoebas

c. Foraminifera d. Diatoms

A P P LY 1. Analyze the following statements and choose the one that most accurately supports the endosymbiotic theory. a. b.

Mitochondria rely on mitosis for replication. Chloroplasts contain DNA but translation does not occur in chloroplasts.

c. d.

Vacuoles have double membranes. Antibiotics that inhibit protein synthesis in bacteria can have the same effect on mitochondria.

2. Which feature of the choanoflagellates was likely the most significant for the evolution of animals? a. b. c. d.

Flagellum with a funnel-shaped, contractile collar also found in sponges A tyrosine kinase receptor on the surface of choanoflagellates that has strong homology to fungi A colonial form that resembles some fungi Eyespots that are similar to those of ribbon worms

3. Examine the life cycle of cellular slime molds, and determine which feature affords the greatest advantage for surviving food shortages. a. b. c. d.

Cellular slime molds produce spores when starved. Cellular slime molds are saprobes. A diet of bacteria ensures there will never be a shortage of food. Cellular slime molds use cAMP to guide each other to food sources.

SYNTHESIZE 1. Modern taxonomic treatments rely heavily on phylogenetic data to classify organisms. In the past, taxonomists often used a morphological species concept, in which species were defined based on similarities in growth form. Give an example of how a morphological species concept would group a set of protists differently than a phylogenetic species concept would. 2. Three methods have been used to try to eradicate malaria. One is to eliminate the mosquito vectors of the parasite, a second is to kill the parasites after they have entered the human body, and the third is to develop a vaccine against the parasite, allowing the human immune system to provide protection from the disease. Which do you suppose is the most promising in the long run? Why? Think about both the biology of the disease and the efficacy of carrying out each of the methods on a large scale. 3. Design an experiment to demonstrate that cells of cellular slime molds are attracted to cyclic AMP. Then, design a follow-up experiment to determine whether they are attracted to cAMP at all times, or only when resources are scarce.

chapter

28 Protists 609

29 CHAPTER

Seedless Plants Chapter Contents 29.1

Origin of Land Plants

29.2 Bryophytes Have a Dominant Gametophyte Generation 29.3 Tracheophytes Have a Dominant Sporophyte Generation 29.4 Lycophytes Diverged from the Main Lineage of Vascular Plants 29.5 Pterophytes Are the Ferns and Their Relatives

©Doug Sherman/Geofile

Introduction

Visual Outline Freshwater green algae

evolved from

Land plants

Haplodiplontic life cycles

have

are classified as

Bryophytes (nontracheophytes)

Tracheophytes

which include

are classified as

Liverworts

Mosses

Hornworts

Seedless plants

Seed plants

which are the

which are the

Lycophytes

Ferns + Allies

Gymnosperms

Angiosperms

The colonization of land by plants fundamentally altered the history of life on Earth. A terrestrial environment offers abundant CO2 and solar radiation for photosynthesis. But for at least 500 million years, the lack of water and the higher ultraviolet (UV) radiation on land confined green algal ancestors of plants to an aquatic environment. Evolutionary innovations for reproduction, structural support, and prevention of water loss are key in the story of plant adaptation to land. The evolutionary shift on land to life cycles dominated by a diploid generation protects against potentially damaging mutations arising from higher UV exposure that could be lethal to an organism with a predominantly haploid generation. As a result, larger numbers of alleles persist in the gene pool, creating greater genetic diversity. Long before seeds and flowers evolved, the seedless plants (like the moss that covers this tree and the

sword ferns that grow alongside it) covered the Earth. In this chapter we consider the evolutionary innovations in seedless plants during the first 100 million years of terrestrial life.

29.1 Origin

of Land Plants

Learning Outcomes 1. Explain the relationship between the different algae clades and plants. 2. Describe the haplodiplontic life cycle. 3. Distinguish between a sporophyte and a gametophyte. 4. Identify two major environmental challenges for land plants and associated adaptations.

Five hundred million years ago, most of the continents were in the southern hemisphere and a diverse array of animals swam through the seas, feeding on an abundance of algae. In stark contrast, the terrestrial environment was a barren landscape. Slowly, sea life began to explore the possibilities of living on land. Eventually, land plants evolved from green algae. This began the conversion of bare ground to the rich and diverse terrestrial ecosystems of today. Green algae and the land plants shared a common ancestor a little over 1 bya and are collectively referred to as the green plants. DNA sequence data are consistent with the claim that a single individual gave rise to all green plants. The green plants are Green plants

Streptophyta

photoautotrophic, but not all photoautotrophs are plants, as some microorganisms are capable of photoautotrophy. Although the plant kingdom includes the green algae, it does not include the fungi, which are more closely related to metazoan animals (see chapter 32). Fungi, however, were essential to the colonization of land by plants, enhancing plants’ nutrient uptake from the soil. One of the most significant evolutionary events in the billion-year-old history of the green plants is the adaptation to terrestrial living. Thus, this chapter and chapter 30 focus on the land plants. Land plants had to develop mechanisms to avoid water loss, to protect from the harmful effects of solar radiation, and to effectively disseminate gametes for reproduction. In addition, although all land plants alternate between haploid gametophyte and diploid sporophyte generations, there is a trend toward dominance of the sporophyte in the more advanced groups.

Land plants evolved from freshwater algae Some saltwater algae evolved to thrive in freshwater environments. Just a single species of freshwater green algae gave rise to the entire terrestrial plant lineage, from mosses through the flowering plants (angiosperms). Exactly what this ancestral alga was is still a mystery, but close relatives, members of the charophytes, exist in freshwater lakes today. The green algae split into two major groups: the chlorophytes, which never made it to land, and the charophytes, which are a sister clade to all the land plants (figure 29.1). Together char­ ophytes and land plants are referred to as streptophytes. Land plants have multicellular haploid and diploid stages, unlike the

Land plants Bryophytes

Red Algae

Green algae

Green algae

Chlorophytes

Charophytes

Liverworts

Tracheophytes

Mosses

Hornworts

Lycophytes

Euphyllophytes Ferns + Allies

Seed plants Gymnosperms

Angiosperms

Ancestral alga

Figure 29.1 Green plant phylogeny. chapter

29 Seedless Plants 611

charophytes. The presence of a diploid embryo in the life cycle is also an innovation of the land plants. As land plants have evolved, the trend has been toward more embryo protection and a reduced haploid stage in the life cycle.

Germination and mitotic cell division

The haplodiplontic life cycle alternates haploid and diploid generations Humans and other animals have a diplontic life cycle, meaning that only the diploid stage is multicellular. In animals, meiosis produces single-celled haploid gametes (egg and sperm cells). These gametes fuse at fertilization to produce a diploid embryo, which divides by mitosis to become the next generation. The products of meiosis never divide by mitosis, so there is no multicellular haploid generation in animals. By contrast, the land plant life cycle is haplodiplontic: It has both multicellular haploid and diploid stages. Land plants undergo mitosis after both fertilization (the diploid stage) and meiosis (the haploid stage). The result is a multicellular diploid individual (called the sporophyte) and a multicellular haploid individual (called the gametophyte). The haplodiplontic cycle is a fundamental feature of plants and is summarized in f­ igure 29.2. Many brown, red, and green algae are also haplodiplontic. Humans produce gametes via meiosis, but land plants actually produce gametes by mitosis in a multicellular, haploid individ­ual. Meiosis produces a haploid spore that divides by mitosis to produce a haploid gametophyte. The multicellular diploid generation, or sporophyte, alternates with the multicellular haploid generation, or gametophyte. Sporophyte means “spore plant,” and gametophyte means “gamete plant.” These terms indicate the types of reproductive cells the respective generations produce. 612 part V Diversity of Life on Earth

MITOSIS

Spore

Land plants are adapted to terrestrial life Unlike their freshwater ancestors, most land plants are not immersed in water. As an adaptation to living on land, most plants are protected from drying out by a waxy surface material called the cuticle that is secreted onto parts that are exposed to the air. The cuticle is relatively impermeable, preventing water loss. This solution, however, limits the gas exchange essential for respiration and photosynthesis. Tiny mouth-shaped openings. called stomata (singular, stoma) in the cuticle of land plants allow the absorption of carbon dioxide and, simultaneously, the release of oxygen and water vapor. Chapter 36 describes how stomata can be closed at times to limit water loss. Moving water within plants is a challenge that increases with plant size. Bryophytes, the most primitive of the land plants (figure  29.1), cannot grow beyond a certain height because they lack vasculature to efficiently transport water. Tracheophytes are more advanced, producing specialized vascular tissue for transport over long distances: xylem is the tissue that carries water; dissolved nutrients, including sugars produced by photosynthesis, are carried in phloem (see chapter 36). Terrestrial plants are exposed to higher intensities of UV irradiation than aquatic algae, increasing the chance of mutation. These land plants minimize the effects of mutation by carrying two copies of every gene. That is, their bodies are diploid, meaning that deleterious recessive mutations are masked. All land plants have both haploid and diploid generations, but there is an evolutionary shift toward a dominant diploid generation.

Gametophyte (n)

n

Sperm

n

n n

Egg Spores

MEIOSIS

n

FERTILIZATION

2n

2n Spore mother cell

2n Zygote

Sporangia

2n

Embryo

Sporophyte (2n)

Figure 29.2 A generalized haplodiplontic plant life cycle. Note that both haploid and diploid individuals are

multicellular. Also, spores are produced by meiosis, but gametes are produced by mitosis.

The diploid sporophyte produces haploid spores (not gametes) by meiosis. Meiosis takes place in structures called sporangia, where diploid spore mother cells (sporocytes) undergo meiosis, each producing four haploid spores. Spores are the first cells of the gametophyte generation. Spores germinate and divide by mitotic cell division to produce a multicellular, haploid gametophyte. The haploid gametophyte produces gametes by mitotic cell division. Consequently, the gametes (egg cells and sperm cells) are also haploid. When two gametes fuse, the zygote they form is diploid and is the first cell of the next sporophyte generation. The zygote grows into a diploid sporophyte by mitosis and produces sporangia, in which meiotic cell division ultimately occurs.

The relative sizes of haploid and diploid generations vary All land plants are haplodiplontic; however, the haploid generation consumes a much larger portion of the life cycle in mosses and ferns than it does in the seed plants—the gymnosperms and angiosperms. In mosses and ferns, the gametophyte is photosynthetic and free-living. When you see moss, you are actually looking at the gametophyte (haploid) generation. The sporophyte is a small structure attached to the gametophyte. Although ferns produce photosynthetic gametophytes, they are barely visible to the naked eye. When you see a fern, you are looking at the sporophyte generation. The gametophyte is even smaller in the seed plants, typically being microscopic. So, as with the ferns, the sporophyte in the seed plants is the dominant generation. Although the sporophyte generation can get very large, the size of the gametophyte is limited in all plants. When the gametophyte generation of mosses produces gametes at its tips, the egg

remains stationary, whereas the sperm swims to the egg in a droplet of water. In the more advanced plants, the tracheophytes, the gametophyte is composed of only a few cells and the sperm no longer need to swim to reach the egg cell. Having completed an overview of plant life cycles, we next consider the major groups of seedless land plants. As we proceed, you will see a reduction of the size of the gametophyte and gametangia (structures in which gametes are produced), with a corresponding increase in specialization for life on land.

Learning Outcomes Review 29.1

A single freshwater green alga successfully invaded land, establishing the land plants. Land plants developed reproductive strategies, conducting systems, stomata, and cuticles as adaptations to terrestrial life. Green plants include the green algae and the land plants, whereas the streptophytes include only the land plants and their sister clade, the charophytes. Most plants have a haplodiplontic life cycle in which a haploid form alternates with a diploid form in a single organism. Diploid sporophytes produce haploid spores by meiosis. Each spore develops into a haploid gametophyte by mitotic cell division; the gametophyte produces haploid gametes, again by mitosis. When the gametes fuse, the diploid sporophyte is formed once more. ■■ ■■

How would you distinguish a small aquatic tracheophyte from a freshwater alga? What distinguishes gamete formation in plants from gamete formation in humans?

29.2 Bryophytes

Have a Dominant Gametophyte Generation

Learning Outcome

Tracheophytes

Hornworts

Mosses

Liverworts

Bryophytes are the closest living descendants of the first land plants. Plants in this group are also called nontracheophytes because they lack the transport cells called tracheids. Bryophytes include the liverworts (phylum Hepaticophyta), the mosses (phylum Bryophyta), and the hornworts (phylum Anthocerotophyta).

Charophytes

1. Describe adaptations of bryophytes for life on land.

Water and gas availability were limiting factors for early terrestrial plants. These plants likely had little ability to regulate internal water levels and tolerated drying out, as is seen in most modern (extant) mosses. Fungi and early land plants cohabited, and the fungi formed close associations with the plants, enhancing water uptake. The beneficial symbiotic relationships between fungi and plants, called mycorrhizal associations, are also found in many existing bryophytes. More information on mycorrhizal fungi is found in chapter 31.

Bryophytes have a photosynthetic gametophyte The approximately 16,000 species of bryophytes are simple but are found in diverse terrestrial environments, even deserts. The gametophyte is small but specialized for light capture and photosynthesis. Water and nutrients are transported throughout the gametophyte via simple conducting cells. Egg and sperm cells produced by the gametophyte unite to form the sporophyte generation, which typically grows upright from the surface of the ground-dwelling gametophyte. Because the sporophyte is above the ground, it can effectively disperse the spores it makes. These spores are carried by air currents and when they land, they germinate into new gametophyte plants. Scientists now agree that bryophytes consist of three quite distinct clades of relatively unspecialized plants: liverworts, mosses, and hornworts. Like their aquatic ancestors, bryophytes require water (such as rainwater) to reproduce sexually. This is also seen in ferns and some other vascular plants (tracheophytes). The sperm must swim to the egg. It is not surprising that bryophytes are especially common in moist places, in both the tropics and temperate regions. They are commonly found on damp forest floors and by stream beds.

Liverworts are an ancient phylum The Old English word wyrt means “plant” or “herb.” Some common liverworts have flattened gametophytes with lobes resembling those of the liver—hence the name “liverwort.” Although the lobed liverworts are the best-known representatives of this phylum, they constitute only about 20% of the species (figure 29.3). The other 80% are leafy and superficially resemble mosses. The gametophytes are flat instead of upright, with single-celled rhizoids that aid in absorption. Although rhizoids function like roots, they are not organs because they are unicellular. Some liverworts have air chambers containing rows of photosynthetic cells, each chamber having a pore at the top to facilitate gas exchange. Unlike stomata, the pores are always open and cannot be closed. Sexual reproduction in liverworts is similar to that in mosses. Lobed liverworts may form gametangia in umbrella-like structures. In this case, the sporophyte is suspended above the ground by the gametophyte. This is different from most bryophytes, in which the sporophyte itself forms a stalk above the ground to aid in spore dispersal. Asexual reproduction occurs when lens-shaped pieces of tissue that are released from the gametophyte grow to form new gametophytes. chapter

29 Seedless Plants 613

Female gametophyte

runs lengthwise down the middle. Only one cell layer thick (except at the midrib), they lack vascular strands and stomata, and all the cells are haploid. However, mosses do have stomata on the capsule portion of the sporophyte and because of that are the basal land group with stomata. Although mosses lack a vascular system, water may be passively carried up a strand of specialized cells in the center of a moss gametophyte. Some mosses also have specialized foodconducting cells surrounding those that conduct water.

Moss reproduction

Figure 29.3 A common liverwort, Marchantia (phylum Hepaticophyta). The microscopic sporophytes are formed by fertilization within the tissues of the umbrella-shaped structures that arise from the surface of the flat, green, creeping gametophyte.

Steven P. Lynch

Mosses have rhizoids and water-conducting tissue

Multicellular gametangia (gamete-producing structures) are formed at the tips of the leafy moss gametophytes (figure 29.5). Female gametangia (archegonia) may develop either on the same gametophyte as the male gametangia (antheridia) or on separate plants, depending on the species. A single egg is produced in the swollen lower part of an archegonium, whereas numerous flagellated sperm are produced in an antheridium. Because gametophytes are haploid, eggs and sperm are produced by mitotic cell division (not meiotic cell division) in the gametangia.

Unlike other bryophytes, the gametophytes of mosses typically consist of small, leaflike photosynthetic structures (not true leaves, which contain vascular tissue) arranged around a stemlike axis (figure 29.4); the axis is anchored to its substrate by means of rhizoids. Each rhizoid consists of several cells that absorb water, but not nearly the volume of water that is absorbed by a vascular plant root. Moss leaflike structures have little in common with leaves of vascular plants, except for the superficial appearance of the green, photosynthetic, flattened blade and slightly thickened midrib that

Sperm

Gametophytes

Male

Egg

Antheridia

Archegonia Germinating spores

Female Rhizoids

FE

RT

I

Z LI

I AT

ON

Zygote n

2n

MITOSIS

MITOSIS

Spores SI O EI

Developing sporophyte in archegonium

M

Gametophyte

2n

Sporangium Parent gametophyte

Figure 29.4 A hair-cup moss, Polytrichum (phylum Bryophyta). The leaflike structures belong to the gametophyte.

Each of the yellowish brown stalks with a capsule, or sporangium, at its summit is a sporophyte. wayra/Getty Images 614 part V Diversity of Life on Earth

2n n

Mature sporophyte

S

Sporophyte

n

Figure 29.5 Life cycle of a typical moss. The majority of the

life cycle of a moss is in the haploid state. The leafy gametophyte (n) is photosynthetic, but the sporophyte (2n) is not and is nutritionally dependent on the gametophyte. Water is required to carry sperm to the egg.

When sperm are released from an antheridium, they swim with the aid of flagella through a film of dew or rainwater to the archegonia. One sperm (which is haploid) unites with an egg (also haploid), forming a diploid zygote. The zygote divides by mitotic cell division and develops into the sporophyte, a slender stalk with a swollen capsule, the sporangium, at its tip. The base of the sporophyte is embedded in gametophyte tissue, its nutritional source. The sporangium is often cylindrical or club-shaped. Spore mother cells within the sporangium undergo meiosis, each producing four genetically different haploid spores. In many mosses at maturity, the top of the sporangium pops off, and the spores are released. A spore that lands in a suitable damp location may germinate and grow, via miotitic cell division into a threadlike structure, which branches to form rhizoids and “buds” that grow upright. Each bud develops into a new leafy gametophyte plant.

Moss distribution In the Arctic and the Antarctic, mosses are the most abundant plants. The greatest diversity of moss species, however, is found in the tropics. Many mosses are able to withstand prolonged periods of drought, although mosses are not common in deserts. Most mosses are highly sensitive to air pollution and are rarely found in abundance in or near cities or other areas with poor air quality. Some mosses, such as the peat mosses (Sphagnum), can absorb up to 25 times their weight in water and are valuable commercially in potting soils or as a fuel when dry.

The moss genome Moss plants can survive extreme water loss—an adaptive trait in the early colonization of land. Desiccation tolerance and phylogenetic position convinced researchers to choose the moss Physcomitrella patens as the first nontracheophyte plant to have its genome sequenced. DNA sequence data reveal that genes associated with an aquatic life were lost in the earliest common ancestor of the land plants. Genes associated with tolerance of terrestrial stresses, including temperature and water availability, are absent in the green alga Chlamy­domonas, but present in moss.

Hornworts are the sister group to tracheophytes The origin of hornworts (phylum Anthocerotophyta) is a puzzle. They are most likely among the earliest land plants, yet the earliest hornwort fossil spores date from the Cretaceous period (65 to 145 mya), when angiosperms were emerging. There are only about 200 species of hornwort; however, the hornworts form an important link in the study of plant evolution because they are the sister group to the tracheophytes. The hornworts are likely to provide insights into the transition from the dominant gametophyte generation in the bryophtyes to the dominant sporophyte generation in the tracheophytes. The sporophytes of the hornworts are most similar to vascular plant sporophytes. Hornwort sporophytes look like green horns, which rise from a flattened structure called a thallus that is usually 1 to 2 cm in diameter (figure 29.6). The sporophyte base is embedded in gametophyte tissue, from which it derives some of its nutrition. It has stomata to regulate gas exchange, is photosyn­thetic, and provides much of the energy needed for growth and reproduction. Meiotic cell division occurs within the “horn” and spores are released along

Photosynthetic sporophyte

Figure 29.6 Hornworts (phylum Anthocerotophyta). 

Hornwort sporophytes are seen in this photo. Unlike the sporophytes of other bryophytes, most hornwort sporophytes are photosynthetic. Steven P. Lynch

its entire length. Hornwort cells usually have a single large chloroplast similar to that found in algae. Hornwort gametophytes typically live in symbiotic association with cyanobacteria, which provide them with nitrogen (see chapter 27).

Learning Outcomes Review 29.2

The bryophytes exhibit adaptations to terrestrial life. Moss adaptations include rhizoids to anchor the moss body and to absorb water, and water-conducting tissues. Mosses are found in a variety of habitats, and some can survive droughts. Liverworts are the oldest bryophyte phylum, whereas hornworts are the closest relatives to the tracheophytes. ■■

What might account for the abundance of mosses in the Arctic and Antarctic?

29.3 Tracheophytes

Have a Dominant Sporophyte Generation

Learning Outcomes 1. Explain the evolutionary significance of tracheids. 2. Evaluate the significance of roots, stems, and leaves in the tracheophytes.

Tracheophytes, also known as vascular plants, first appeared about 410 mya. The first tracheophytes with a relatively complete record belonged to the phylum Rhyniophyta. It is impossible to know for sure what the earliest of these vascular plants looked like, but fossils of Cooksonia provide some insight into their characteristics (figure 29.7). chapter

29 Seedless Plants 615

Sporangia

the sporophyte is the dominant generation in the tracheophytes. A green moss plant is a gametophyte, whereas a green tree, for example, is a sporophyte. (Vascular tissue structure is discussed more fully in chapter 36.) Other features found in all vascular plant sporophytes include a waxy cuticle on above-ground parts and pores called stomata. Both features reduce water loss by land plants.

?

Inquiry question Explain why tracheophytes may

have had a selective advantage over bryophytes during the evolution of land plants.

Tracheophytes are grouped in three clades Figure 29.7 Cooksonia, the first known vascular land plant. This fossil represents a plant that lived some 420 mya. Cooksonia

belongs to phylum Rhyniophyta, consisting entirely of extinct plants. Its upright, branched stems, which were no more than a few centimeters tall, terminated in sporangia, as seen here. It probably lived in moist environments such as mudflats, had a water-resistant cuticle, and produced spores typical of vascular plants. The Natural History Museum/Alamy Stock Photo

Cooksonia, the first known vascular land plant, appeared in the late Silurian period about 420 mya, but is now extinct. It was successful partly because it encountered little competition as it spread out over vast tracts of barren land. The plants were only a few centimeters tall and had no roots or leaves. They consisted of little more than a branching stemlike structure, with the branches forking evenly and expanding slightly toward the tips. They were ­homosporous (producing only one type of spore). Sporangia formed at branch tips. Other ancient vascular plants that followed evolved more complex arrangements of sporangia. A major feature of the tracheophytes is the dominant sporophyte generation. We do not yet know how the transition from the dominant gametophyte in the bryophytes to the dominant sporophyte in the tracheophytes occurred. However, the evolutionary advantage of having two sets of chromosomes in the conspicuous generation is significant, as discussed in section 29.1.

Three clades of vascular plants exist today: (1) lycophytes (club mosses), (2) pterophytes (ferns and their relatives), and (3) seed plants. The first two clades are explored in this chapter (table 29.1). Advances in molecular systematics have changed the way we view the evolutionary history of vascular plants. Whisk ferns and horsetails were long believed to be distinct phyla that were transitional between bryophytes and vascular plants. Phylogenetic evidence now shows they are the closest living relatives to ferns, and they are grouped as pterophytes. Tracheophytes dominate terrestrial habitats everywhere, except for the highest mountains and the tundra. The haplodiplontic life cycle persists, but the gametophyte has been reduced in size relative to the sporophyte during the evolution of tracheophytes. A similar reduction in multicellular gametangia has occurred as well. The reduction in the size of the gametophyte is a major theme in the evolution of land plants.

Stems evolved prior to roots Fossils of early vascular plants reveal stems, but no roots or leaves. The earliest vascular plants, including Cooksonia, had transport cells in their stems, but the lack of roots limited the size of these plants in two ways. First, roots anchor plants to the ground, preventing them from falling over. Tall plants need strong roots. Second, a large plant has a large surface area for water loss. An extensive root system is needed to ensure the plant’s need for water is met.

Vascular tissue allows for distribution of nutrients

Roots provide structural support and transport capability

Cooksonia and the other early tracheophytes became successful colonizers of the land by developing efficient water- and foodconducting systems called vascular tissues. These tissues consist of strands of specialized cells that form a network throughout a plant, extending from the roots, through the stems, and into the leaves. One type of vascular tissue, xylem, conducts water and dissolved minerals upward from the roots; another type of tissue, phloem, conducts sucrose and hormones throughout the plant. In the early vascular plants, tracheids were the only cells that conducted water in xylem tissue. Later, plants developed even more effective water transport cells, called vessel elements. Vascular tissue allows tracheophytes to achieve great size, like the coastal redwood that can grow to over 300 feet high. Vascular tissue develops in the sporophyte, but (with a few exceptions) not in the gametophyte. It is important to note that

True roots are found only in the tracheophytes. Other, somewhat similar structures enhance either transport or support in nontracheophytes, but only roots have a dual function: providing both transport and support. Lycophytes diverged from other tracheophytes before roots appeared, based on fossil evidence. It appears that roots evolved at least twice.

616 part V Diversity of Life on Earth

Leaves evolved more than once Leaves increase surface area of the sporophyte, enhancing photosynthetic capacity. Lycophytes have single vascular strands supporting relatively small leaves called lycophylls. True leaves, called euphylls, are found only in ferns and seed plants, having distinct origins from lycophylls (­figure 29.8). Lycophylls may have

TA B L E 2 9 .1

The Phyla of Extant Seedless Vascular Plants

Phylum

Examples

Approximate Number of Living Species

Key Characteristics

Lycophyta

Club mosses

Homosporous or heterosporous. Sperm motile. External water necessary for fertilization. About 17 genera.

Pterophyta

Ferns

Primarily homosporous (a few heterosporous). Sperm motile. External water necessary for fertilization. Leaves uncoil as they mature. Sporophytes and virtually all gametophytes are photosynthetic. About 365 genera.

11,000

Horsetails

Homosporous. Sperm motile. External water necessary for fertilization. Stems ribbed, jointed, either photosynthetic or nonphotosynthetic. Leaves scalelike, in whorls; nonphotosynthetic at maturity. One genus.

15

Whisk ferns

Homosporous. Sperm motile. External water necessary for fertilization. No differentiation between root and shoot. No leaves; one of the two genera has scalelike extensions and the other leaflike appendages.

6

Figure 29.8 The evolution of leaves independently in two plant lineages. Lycophylls are small

Lycophyll Origins

Stem with vascular tissue

Stem, leafy tissue without vascular tissue

Stem, leafy tissue with vascular tissue

1275

Single vascular strand (vein)

simple leaves, whereas those of euphylls may be large with a complex system of veins.

Euphyll Origins

Branching stems with vascular tissue

Unequal branching

Branches in single planes

Photosynthetic tissue fills out branches

Branched vascular strands (veins)

chapter

29 Seedless Plants 617

Chlorophytes

Charophytes

Liverworts

Mosses

Hornworts

Lycophytes

Ferns + Allies

Gymnosperms

Angiosperms

Seeds

Euphylls Stems, roots, leaves Dominant sporophyte Vascular tissue

Flowers Fruits

Stomata Multicellular embryo Antheridia and archegonia Cuticle Plasmodesmata Chlorophyll a and b Ancestral alga

Figure 29.9 Land plant innovations.

Learning Outcomes Review 29.3

Most tracheophytes have well-developed vascular tissues, including tracheids, that enable efficient delivery of water and nutrients throughout the organism. They also exhibit specialized roots, stems, leaves, cuticles, and stomata. Many produce seeds, which protect and nourish embryos. ■■

Why would vascular tissue be prevalent in the sporophyte, but not the gametophyte, generation?

618 part V Diversity of Life on Earth

1. Explain features that differentiate lycophytes from bryophytes. 2. Distinguish between lycophytes and other vascular plants.

The earliest vascular plants lacked seeds. Members of four phyla of living vascular plants also lack seeds, as do at least three other phyla known only from fossils. As we explore the adaptations of the vascular plants, we focus on both reproductive strategies and the advantages of increasingly complex transport systems. The lycophytes (club mosses) are the sister group to all other vascular plants (figure 29.10). Despite the early divergence between the lycophytes and all other vascular plants, there are similarities among the groups. For example, leaves developed independently in the lycophytes and other vascular plants. Lycophyte leaves are small unbranched veins, whereas the higher vascular plants produce larger leaves with a complex pattern of branched veins. Some ancient club mosses looked like the trees we see in the seed plants Seed Plants

Seeds are highly resistant structures well suited to protecting the plant embryo from environmental stresses and to some extent from predators. In addition, almost all seeds contain a supply of food for the young plant. Lycophytes and pterophytes do not have seeds. They are dispersed via haploid spores. Fruits in the flowering plants (angiosperms) add a layer of protection to seeds and have adaptations that assist in seed dispersal, expanding the potential range of the species. Flowers allow plants to secure the benefits of wide outcrossing in promoting genetic diversity, as discussed in chapter 30. Before moving on to the specifics of lycophytes and pterophytes, review the evolutionary history of terrestrial innovations in the land plants illustrated in figure 29.9.

Learning Outcomes

Ferns and Allies

Seeds are another innovation in the advanced tracheophyte phyla

Diverged from the Main Lineage of Vascular Plants

Lycophytes

Data analysis Compare the amount of time that passed between the early diversification of land plants, the origins of tracheophytes, and the origins of leaves.

29.4 Lycophytes

Hornworts

resulted from vascular tissue penetrating small, leafy protuberances on stems. Euphylls most likely arose from branching stems that became webbed with leaf tissue.

Figure 29.10  A club moss. Selaginella

moellendorffii’s sporophyte generation grows on moist forest floors. Dr. Jody Banks,

Purdue University

Learning Outcomes Review 29.4

Lycophytes are ancestral to all other vascular plants. Although they superficially resemble bryophytes, they have a dominant sporophyte generation. They contain tracheid-based vascular tissues and their reproductive cycle is like that of other vascular plants; however, their leaves are simple, with unbranched veins. ■■

What factors may have led to the independent origins of similar growth forms in the lycophytes and the other vascular plants?

29.5 Pterophytes

Are the Ferns and Their Relatives

Learning Outcomes 1. List the features exhibited by pterophytes. 2. Contrast pterophyte and moss sporophytes.

Seed Plants

Whisk Ferns

Ferns

Horsetail Ferns

Ferns

The phylogenetic relationships among ferns and their near relations is intriguing. A common ancestor gave rise to two clades: one clade diverged to produce a line of ferns and horsetails; the other diverged to yield another line of ferns and whisk ferns—ancient-looking plants. Whisk ferns and horsetails are close relatives of ferns. Like lycophytes and bryophytes, they all form antheridia (containing sperm) and archegonia (containing eggs). Water is required for the process of fertilization, during which the sperm, which have flagella, swim to and unite with the eggs. In contrast, most seed plants have nonflagellated sperm. Lycophytes

of today. The treelike growth form is another example of independent origins of similar features in the lycophytes and the other vascular plants. Today, club mosses are worldwide in distribution but are most abundant in the tropics and moist temperate regions. However, modern club mosses are small. None have the treelike growth form of their ancient ancestors. Club mosses superficially resemble true mosses, but once their internal vascular structure and reproductive processes became known, it was clear that they are unrelated to mosses. The sporophyte stage is the dominant (obvious) stage. Specialized leaves bear spores, which are produced by meiosis. In most lycophytes, each haploid spore germinates into a small free-living gametophyte that produces both sperm and egg cells. The gametophyte of some lycophytes is photosynthetic, whereas that of other species grows below ground and obtains its nourishment from a symbiotic association with soil fungi. After fertilization, the small sporophyte remains nutritionally dependent on the gametophyte until its green leaves develop and start to carry out photosynthesis. The lycophyte Selaginella moellendorffii was the first seedless plant to have its genome fully sequenced. A few clues to the evolution of vascular plants, hidden in the genome, emerged in comparisons with genomes of flowering plants. They supported the independent origins of leaflike structures in different vascular plant lineages. Genome differences also reflect differences in developmental pathways leading to reproductive maturity in the sporophyte generation in lycophytes and flowering plants. Comparing predicted proteins in the Chlamydomonas (green alga), Physcomitrella (moss), and Selaginella with 15 angiosperms revealed 3814 gene families that all the green plants share—the essential instructions for building a green plant. About 3000 new genes were needed in the transition from the single-celled green alga to the multicellular moss, but only 516 genes were needed for the transition from nonvascular to vascular plants. This is a first step in sorting out the evolutionary steps that led to the vascular plants.

Whisk ferns lack roots and leaves In whisk ferns, which occur in the tropics and subtropics, the sporophytic generation consists merely of evenly forking green stems without roots (figure 29.11). The two or three species of the genus Psilotum do, however, have tiny, green, spirally arranged flaps of leaflike tissue lacking veins and stomata. Another genus, Tmesipteris, has appendages that look even more like leaves. Currently, systematists believe that whisk ferns lost their leaves and roots when they diverged from others in the fern lineage. Given the simple structure of whisk ferns, it was particularly surprising to discover that they are monophyletic with ferns. The gametophytes of whisk ferns are essentially colorless and chapter

29 Seedless Plants 619

Figure 29.11  A whisk fern. 

Whisk ferns have no roots or leaves. The green, photosynthetic stems have yellow sporangia attached. Meiotic cell division in the sporangia produces haploid spores, which are released and germinate into gametophytes. Kingsley R. Stern

Horsetail sporophytes consist of ribbed, jointed, photosynthetic stems that arise from branching underground rhizomes with roots at their nodes. A whorl of nonphotosynthetic, scalelike leaves emerges at each node. The hollow stems have silica deposits in the epidermal cells. Horsetails are also called scouring rushes because pioneers of the American West used them to scrub pans. Conelike structures on horsetail sporophytes produce haploid spores via meiosis. Spores are carried away by air currents and, when they land on the ground, they germinate into photosynthetic gametophtyes. A horsetail gametophyte looks like a small lobed liverwort plant. Each gametophyte produces both eggs and swimming sperm, which unite at fertilization. The young sporophyte will remain dependent on the gametophyte until it produces photosynthetic tissue.

Ferns have fronds that bear sori

very  small. As with some lycophytes, these gametophytes form symbiotic associations with fungi, which furnish their nutrients. Some develop elements of vascular tissue and have the distinction of being the only gametophytes known to do so.

Horsetails have jointed stems with brushlike leaves The 15 living species of horsetails are members of a single genus, Equisetum. Fossil forms of Equisetum extend back 300 million years to an era when some of their relatives were treelike. Today, they are widely scattered around the world, mostly in damp places. Some that grow among the coastal redwoods of California may reach a height of 3 m, but most are less than a meter tall (figure 29.12).

Ferns are the most abundant group of seedless vascular plants, with about 11,000 living species. Recent research indicates that they may be the closest relatives of the seed plants. Rainforests and swamps of fern trees growing in the eastern United States and Europe over 300 mya formed the coal currently being mined. Today, ferns flourish in a wide range of habitats throughout the world; however, about 75% of the species occur in the tropics. The conspicuous sporophytes may be less than a centimeter in diameter (as in small aquatic ferns such as Azolla), or more than 24 m tall, with leaves up to 5 m or longer in the tree ferns (figure 29.13). The sporophytes and the much smaller gametophytes, which rarely reach 6 mm in diameter, are both photosynthetic. The fern life cycle (figure 29.14) differs from that of a moss primarily in the much greater development, independence, and dominance of the fern’s sporophyte. The fern sporophyte is structurally more complex than the moss sporophyte, having vascular tissue and well-differentiated roots, stems, and leaves. The gametophyte, however, lacks the vascular tissue found in the sporophyte.

Fern morphology Figure 29.12  A horsetail, Equisetum telmateia. This species

forms two kinds of erect stems; one is green and photosynthetic, and the other, which terminates in a spore-producing “cone,” is mostly light brown. Stephen

P. Parker/Science Source

Fern sporophytes, like horsetails, have rhizomes (horizontal underground stems). Leaves, referred to as fronds, usually develop at the tip of the rhizome as tightly rolled-up coils (“fiddleheads”) that unroll and expand (figure 29.15). The tight coil of the fiddlehead

Figure 29.13  A tree fern (phylum Pterophyta) in the forests of Malaysia.  The ferns are by far the largest group of seedless vascular plants. Bildagentur Zoonar GmbH/Shutterstock

620 part V Diversity of Life on Earth

Figure 29.14  Life cycle of a typical fern. Both the

gametophyte (n) and sporophyte (2n) are photosynthetic and can live independently. Water is necessary for fertilization. Sperm are released on the underside of the gametophyte and swim in moist soil to neighboring gametophytes. Spores are dispersed by wind.

Antheridium

Archegonium

Rhizoids

Archegonium Egg

Sperm

Gametophyte MITOSIS

Antheridium

Spores

FE

R

I TIL

TIO ZA

N

n

MEIOSIS

Zygote 2n

n MITOSIS

2n

Underside of leaf frond

Mature frond

Adult sporophyte

Mature sporangium

Leaf of young sporophyte

Sorus (cluster of sporangia)

Embryo

Gametophyte

Sporangium

Rhizome

Tightly Coiled Fern

Uncoiling Fern

Figure 29.15 Fern “fiddlehead.” Fronds develop in a coil

and slowly unfold on ferns, including the tree fern fronds in these photos. (left) Ingram Publishing/Alamy Stock Photo; (right) Ed Reschke

allows the delicate fern fronds to push out of the soil from their base on underground rhizomes. Once in the air, the fronds can unfurl without being damaged. Fiddleheads are considered a delicacy in several cuisines, but some species contain secondary compounds linked to stomach cancer. Many fronds are highly dissected and feathery, making the ferns that produce them prized as ornamental garden plants. Some ferns, such as Marsilea, have fronds that resemble a fourleaf clover, but Marsilea fronds still begin as coiled fiddleheads. Other ferns produce a mixture of photosynthetic fronds and nonphotosynthetic reproductive fronds that tend to be brownish in color.

Fern reproduction Ferns produce distinctive sporangia, usually in clusters called sori (singular, sorus), typically on the underside of the fronds. Sori are chapter

29 Seedless Plants 621

often protected during their development by a transparent, umbrella-like covering. (At first glance, one might mistake the sori for an infection on the plant.) Diploid spore mother cells in each sporangium undergo meiosis, producing haploid spores. At maturity, the spores are catapulted from the sporangium by a snapping action, and those that land in suitable damp locations may germinate, producing photosynthetic gametophytes that are often heart-shaped, are only one cell layer thick (except in the center), and have rhizoids that anchor them to their substrate. These rhizoids are not true roots because they lack vascular tissue, but they do aid in transporting water and nutrients from the soil. Flask-shaped archegonia and globular antheridia are produced on either the same or a different gametophyte. The multicellular archegonia provide some protection for the developing embryo. The sperm formed in the antheridia have flagella, with which they swim toward the archegonia when water is present, often in response to a chemical signal secreted by the archegonia. One

sperm unites with the single egg toward the base of an archegonium, forming a zygote. The zygote then develops into a new sporophyte, completing the life cycle (see figure 29.14). The developing fern embryo has substantially more protection from the environment than a charophyte zygote, but it cannot enter a dormant phase to survive a harsh winter the way a seed plant embryo can.

Learning Outcomes Review 29.5

Ferns and their relatives have a large and conspicuous sporophyte with vascular tissue. Many have well-differentiated roots, stems, and leaves (fronds). The gametophyte generation is small and green, but lacks vascular tissue. ■■

Why are the fiddleheads of some fern species poisonous?

Chapter Review ©Doug Sherman/Geofile

29.1 Origin of Land Plants Land plants evolved from freshwater algae. Green algae and the land plants are called green plants. All green plants arose from a single freshwater green algal species (figure 29.1). The charophytes are the sister clade of the land plants and together these groups are called the streptophytes. Land plants are adapted to terrestrial life. Land plants have two major characteristics: protected embryos and multicellular haploid and diploid phases. A waxy cuticle, stomata, and specialized cells for transport of water and minerals enhance survival. The haplodiplontic life cycle alternates haploid and diploid generations. Most multicellular green plants have haplodiplontic life cycles. Plants have a haplodiplontic life cycle with multicellular diploid sporophytes and haploid gametophytes (figure 29.2). The relative sizes of haploid and diploid generations vary. As some plants became more complex, the sporophyte stage became the dominant phase.

29.2 Bryophytes Have a Dominant Gametophyte

Generation

Bryophytes have a photosynthetic gametophyte. Bryophytes consist of three distinct clades: liverworts, mosses, and hornworts. Bryophytes do not have true roots or tracheids, but do have conducting cells for movement of water and nutrients. In liverworts and mosses, the nonphotosynthetic sporophyte is nutritionally dependent on the gametophyte. 622 part V Diversity of Life on Earth

Liverworts are an ancient phylum. The gametophyte of some liverworts is flattened and has lobes that resemble those of the liver. They produce upright structures that contain the gametangia. Mosses have rhizoids and water-conducting tissue. Mosses exhibit alternation of generations and have widespread distribution. Many are able to withstand droughts. Hornworts are the sister group to tracheophytes. Stomata in the sporophyte can open and close to regulate gas exchange. The sporophyte is also photosynthetic.

29.3 Tracheophytes Have a Dominant Sporophyte

Generation (table 29.1)

Vascular tissue allows for distribution of nutrients. The evolution of tracheids allowed more efficient vascular systems to develop. This vascular tissue develops in the sporophyte. Vascular plants have a much reduced gametophyte. Tracheophytes are grouped in three clades. The vascular plants found today exist in three clades: lycophytes, pterophytes, and seed plants (figure 29.9). Stems evolved prior to roots. Roots provide structural support and transport capability. Leaves evolved more than once. Lycophytes have small leaves called lycophylls that lack vascularization. True leaves (euphylls) are found only in ferns and seed plants and have origins different from those of lycophylls.

Seeds are another innovation in the advanced tracheophyte phyla. Seeds are resistant structures that protect the embryo from desiccation and to some extent from predators.

29.4 Lycophytes Diverged from the Main Lineage

of Vascular Plants

Lycophyte ancestors were the earliest vascular plants and were among the first plants to have a dominant sporophyte generation.

29.5 Pterophytes Are the Ferns and Their Relatives Ancestors of the pterophytes gave rise to two clades: one line of ferns and horsetails, and a second line of ferns and whisk ferns. Pterophytes require water for fertilization and are seedless.

Whisk ferns lack roots and leaves. The sporophyte of a whisk fern consists of evenly forking green stems without roots. Horsetails have jointed stems with brushlike leaves. Scalelike leaves of horsetail sporophytes emerge in a whorl. The stems have silica deposits in epidermal cells of their ribs. Ferns have fronds that bear sori. The leaves of ferns, called fronds, develop as tightly rolled coils that unwind to expand. Sporangia called sori develop on the underside of the fronds. The gametophyte is often heart shaped and can live independently.

Visual Summary Freshwater green algae

evolved from

divided into

Haplodiplontic life cycle

have

Land plants

mitosis produces

sister clade to

Haploid gametophyte are classified as Chlorophytes

Charophytes

alternates with Diploid sporophyte is dominant in

Bryophytes (nontracheophytes) which have

which have

which have

Tracheophytes

Flattened gametophytes

Water-conducting tissue

Photosynthetic sporophytes

are

are

are

Seed plants

Seedless plants which have

which need Water for fertilization

Hepaticophyta

Bryophyta

Anthocerotophyta

Small, avascular leaves

called

called

called

are

are

Liverworts

Mosses

Hornworts

Lycophytes

Pterophytes

were the

include

Earliest vascular plants

Whisk ferns horsetails ferns

include Gymnosperms Angiosperms

chapter

29 Seedless Plants 623

Review Questions ©Doug Sherman/Geofile

U N D E R S TA N D

c.

1. Which of the following plant structures is NOT matched to its correct function? a. b. c. d.

Stomata—allow gas transfer Tracheids—allow the movement of water and minerals Cuticle—prevents desiccation All of the choices are matched correctly.

2. Which of the following genera most likely directly gave rise to the land plants? a. b.

Volvox Chlamydomonas

c. d.

Ulva Chara

3. Which of the following would NOT be found in a bryophyte? a. b. c. d.

Mycorrhizal associations Rhizoids Tracheid cells Photosynthetic gametophytes

4. Which of the following statements is correct regarding the bryophytes? a. b. c. d.

The bryophytes represent a monophyletic clade. The sporophyte stage of all bryophytes is photosynthetic. Archegonium and antheridium represent haploid structures that produce reproductive cells. Stomata are common to all bryophytes.

5. Evolutionary innovations that increase desiccation tolerance include a. b. c. d.

waxy cuticles. abscisic acid–signaling pathways. rhizoids. All of the choices are correct.

6. Which of the following statements about the pterophytes is accurate? a. b. c. d.

Horsetails and whisk ferns form a single clade. Ferns form a single clade. Whisk ferns have euphylls. All pterophytes have a dominant sporophyte generation.

A P P LY 1. Which of the following correctly compares what happens to a spore mother cell as it gives rise to a spore with what happens to a spore as it gives rise to a gametophyte? a. The spore mother cell and the spore both go through meiosis. b. The spore mother cell and the spore both go through mitosis.

624 part V Diversity of Life on Earth

The spore mother cell goes through mitosis, and the spore goes through meiosis. d. The spore mother cell goes through meiosis, and the spore goes through mitosis. 2. How could a plant without roots obtain sufficient nutrients from the soil? a. b.

It cannot; all land plants have roots. Mycorrhizal fungi associate with the plant and assist with the transfer of nutrients. c. Charophytes associate with the plant and assist with the transfer of nutrients. d. It relies on its xylem in the absence of a root. 3. A major innovation of land plants is embryo protection. How is a moss embryo protected from desiccation? a. b. c. d.

By the seed By the antheridium By the archegonium By the lycophyll

4. The following evolutionary trends are seen in the seedless land plants: a. b.

Gametophytes became photosynthetic. A key innovation accompanying the appearance of pterophytes was the haplodiplontic life cycle. c. The gametophyte generation became the dominant generation. d. There is increased protection of the sporophyte generation in its early developmental stages. 5. Which of the following statements is true? a. b. c. d.

Moss and lycophytes have leaves. Leaves of lycophytes and pterophytes have different origins. Leaves were a bryophyte innovation. Leaf polarity genes in Selaginella support the hypothesis that leaves evolved one time in all the land plants.

SYNTHESIZE 1. You have access to the sequenced genomes for moss and the lycophyte Selaginella. Your goal in analyzing the data is to write a paper that answers an important question about the evolution of land plants. What question would you try to answer? 2. Would you expect the sporophyte generation of a moss or of a fern to have more mitotic divisions? Why? 3. Imagine hypothetical moss and fern trees, each 10 m tall. Which would face the greater barriers to sexual reproduction? Why?

30 CHAPTER

Seed Plants Chapter Contents 30.1

The Evolution of Seed Plants

30.2 Gymnosperms: Plants with “Naked Seeds” 30.3 Angiosperms: The Flowering Plants 30.4 Seeds 30.5 Fruits

Zoonar/O Popova/age fotostock

Introduction

Visual Outline Seed Plants include

Gymnosperms

characterized by

Partially enclosed seeds

Angiosperms

have

Flowers

producing

Enclosed seeds

inside

Fruits

The history of the land plants is replete with evolutionary innovations allowing the ancestors of aquatic algae to colonize the harsh and varied terrestrial terrains. Early innovations made survival on land possible; the subsequent explosion of plant life continues to change the land and atmosphere, and supports terrestrial animal life. Seed-producing plants have come to dominate the terrestrial landscape over the last several hundred million years. Much of the remarkable success of seed plants can be attributed to the evolution of the seed, an innovation, like the beans pictured here, protects delicate embryos and provides food for them. Seeds allow embryos to pause development and growth and germinate after a harsh winter or dry season has passed. Fruits, a later innovation, enhanced the dispersal of embryos across a broader landscape. Within the seed plants, the flowering plants have coevolved intricate relationships with animals to enhance sexual reproduction, as well as aiding dispersal of the next generation.

30.1 The Evolution of Seed Plants Learning Outcomes 1. List the evolutionary advantages of seeds. 2. Distinguish between pollen and sperm in seed plants.

Numerous evolutionary solutions to terrestrial challenges have resulted in over 400,000 species of seed plants dominating all terrestrial communities today. Collectively these seed plants affect almost every aspect of our lives, from improving environmental quality to providing pharmaceuticals, food, fuels, building materials, and clothing. Seed plants appear to have evolved from spore-bearing plants known as progymnosperms. Progymnosperms shared several features with modern gymnosperms, including secondary vascular tissues (which allow for an increase in girth). Some progymnosperms had leaves. Their reproduction was very simple, and it is not certain which group of progymnosperms gave rise to seed plants. Biologists have long been intrigued by the rapid success and diversity of seed plants, particularly the diversification of the flowering plants (angiosperms). Likely the diversity can be attributed to a confluence of features, with the evolution of the seed being one important event. Phylogenetic analysis of seedless and seed plants with sequenced genomes revealed vast numbers of gene duplications around 309 mya, when the early seed plants (ancestral to gymnosperms and angiosperms) began to diversify from their seedless plant ancestor, and again 192 mya when the earliest angiosperms (flowering plants) were appearing. The gene duplications are at such a scale that the most logical explanation is that whole-genome duplications occurred at the origins of both seed plants and angiosperms. As discussed in ­chapter 24, whole-genome duplications can accelerate evolution through mutations in the extra copies of genes.

The seed protects the embryo From an evolutionary and ecological perspective, the seed represents an important advance. The embryo is protected by an extra layer or two of sporophyte tissue called the integument, creating the ovule (figure 30.1). Within the ovule, meiotic cell division occurs in the megasporangium, producing a haploid megaspore. The megaspore

Stored food (endosperm) Integument (seed coat)

divides by mitosis to produce a female gametophyte carrying the female gamete, an egg. The egg combines with the male gamete, a sperm, resulting in the zygote. The single-celled zygote divides by mitotic cell division to produce the young sporophyte, an embryo. Seeds also contain a food supply for the developing embryo. During development, the integuments harden to produce the seed coat, which protects the embryo. The seed is easily dispersed. Perhaps even more significantly, the presence of seeds introduces into the life cycle a dormant phase that allows the embryo to survive until environmental conditions are favorable for further growth.

Water is not required to transport the male gametophyte to the female gametophyte Seed plants produce two kinds of gametophytes—male and female— each of which consists of just a few cells. A consistent feature in the evolution of plants is a reduction in the size of the gametophyte and a corresponding increase in dominance of the sporophyte generation. A pollen grain is a multicellular male gametophyte carrying the male gamete, a sperm cell. Pollen grains are carried to the female gametophyte by wind or by a pollinator. In some seed plants, the sperm moves toward the female gametophyte through a growing pollen tube. This eliminates the need for external water. In contrast to the seedless plants, the whole male gametophyte, rather than just the sperm, moves to the female gametophyte. A female gametophyte forms within the protection of the integuments, collectively forming the ovule. In angiosperms, the ovules are completely enclosed within additional diploid sporophyte tissue. The ovule and the surrounding protective tissue are called the ovary. The ovary develops into the fruit.

Learning Outcomes Review 30.1

The gymnosperms and angiosperms evolved from a common seed-producing ancestor. Whole-genome duplication likely contributed to the rise and dominance of seed plants. Seeds protect the embryo, aid in dispersal, and can allow for an extended pause in the life cycle. Seed plants produce male and female gametophytes; the male gametophyte is a pollen grain, which is carried to the female gametophyte by wind or other means. The male gametophyte carries a sperm cell, whereas the female gametophyte carries an egg cell. ■■

Why is water essential for fertilization in bryophytes and some pterophytes but not in seed plants?

30.2 Gymnosperms:

Plants with “Naked Seeds”

Embryo

Learning Outcomes 300 μm

Figure 30.1 Cross section of an ovule. Biology Media/

Science Source

626 part V Diversity of Life on Earth

1. Describe the distinguishing features of a gymnosperm. 2. List the four groups of living gymnosperms.

TA B L E 3 0 .1

Angiosperms

Gymnosperms

Ferns and Allies

Seeds distinguish the gymnosperms from the pterophytes (ferns and allies). Gymnosperms encompass four of the five lineages of seed plants. The four groups of gymnosperms are the coniferophytes, cycadophytes, gnetophytes, and ­ ginkgophytes. The fifth lineage of seed plants is the angiosperms. Although both gymnosperms and angiosperms produce seeds, only the angiosperms produce flowers and fruits (table 30.1). In gymnosperms, the ovule, which becomes a seed, rests exposed on a scale (a modified shoot or leaf) and is not completely enclosed by sporophyte tissues at the time of pollination. The name gymnosperm means “naked seed” because the seed develops on the surface of the scale. Gymnosperms vary in their reproduction and growth form. For example, cycads and Ginkgo have motile sperm, whereas conifers and gnetophytes have sperm with no flagella. All sperm are carried within a pollen tube that forms when a pollen grain germinates. Most gymnosperms are terrestrial, although a few aquatic species exist.

Conifers are the largest gymnosperm phylum The most familiar gymnosperms are conifers (phylum Coniferophyta), which include pines (figure 30.2), spruces, firs, cedars,

Figure 30.2 Conifers.  Loblolly pine, Pinus taeda, is found on 58 million acres of land in the southeastern part of the United States. Worldwide 16% of the annual timber supply comes from this conifer.  Scott Nodine/Alamy Stock Photo

The Five Phyla of Extant Seed Plants

Phylum

Examples

Key Characteristics

Approximate Number of Living Species

Coniferophyta (sometimes called the Pinophyta)

Conifers (including pines, spruces, firs, yews, redwoods, and others)

Heterosporous seed plants. Sperm not motile; accesses the egg by a pollen tube. Leaves mostly needle-like or scalelike. Trees, shrubs. About 68 genera. Many produce seeds in cones.

630

Cycadophyta

Cycads

Heterosporous. Sperm flagellated and motile but confined within a pollen tube that grows to the vicinity of the egg. Palmlike plants with pinnate leaves. Secondary growth slow compared with that of the conifers. Ten genera. Seeds in cones.

306

Gnetophyta

Gnetophytes

Heterosporous. Sperm not motile; accesses the egg by a pollen tube. The only gymnosperms with vessels in their vasculature. Trees, shrubs, vines. Three very diverse genera (Ephedra, Gnetum, Welwitschia).

65

Ginkgophyta

Ginkgo

Heterosporous. Sperm flagellated and motile but accesses the egg by a pollen tube. Deciduous tree with fan-shaped leaves that have evenly forking veins. Individuals of the species are either male or female. Seeds resemble a small plum with fleshy, foul-smelling outer covering. One genus.

1

Anthophyta

Flowering plants (angiosperms)

Heterosporous. Sperm not motile; accesses the egg by a pollen tube. Seeds enclosed within a fruit. Leaves greatly varied in size and form. Herbs, vines, shrubs, trees. Most significant plant contributor to foods consumed by humans and other animals. About 14,000 genera.

>300,000

chapter

30 Seed Plants 627

hemlocks, yews, larches, cypresses, and others. Nearly 40% of the world’s forests are composed of conifers. The coastal redwood (Sequoia sempervirens), a conifer native to northwestern California and southwestern Oregon, is the tallest living vascular plant; it may reach a height of nearly 100 m (300 ft). Another conifer, the bristlecone pine (Pinus longaeva) of the White Mountains of California, is the oldest living tree; one specimen is 4900 years old. Conifers are found in the colder temperate and sometimes drier regions of the world. Various species are sources of timber, biofuel feedstock, paper, resin, paclitaxel (Taxol, which is used to treat certain cancers), and other economically important products. Genomes of three conifers—loblolly pine, sugar pine, and Douglas fir—have been sequenced because of their economic importance. This was a substantial challenge, because their genomes are about 10 times larger than the human genome.

Pines are an exemplary conifer genus More than 100 species of pines exist today, all native to the northern hemisphere. Pines and spruces, which belong to the same family, are members of the vast coniferous forests that lie between the arctic tundra and the temperate deciduous forests and prairies to their south. During the past century, pines have been extensively planted in the southern hemisphere.

Pine morphology Pines have tough, needle-like leaves produced mostly in clusters of two to five. Among the conifers, only pines have clustered leaves. The leaves, which have a thick cuticle and recessed stomata, represent an evolutionary adaptation for minimizing water loss. This strategy is important because many of the trees grow in areas where the topsoil is frozen for part of the year, making it difficult for the roots to obtain water. The leaves and other parts of the sporophyte have canals into which surrounding cells secrete resin. The resin deters insect and fungal attacks. The resin of certain pines is harvested commercially for its volatile liquid portion, called turpentine, and for the solid rosin, which is used on the bow hair of bowed string instruments. The wood of pines lacks some of the more rigid cell types found in other trees, and it is considered a “soft” rather than a “hard” wood. The thick bark of pines is an adaptation for surviving fires and subzero temperatures. Some cones actually depend on fire to open, releasing seeds to reforest burned areas.

Reproductive structures All seed plants produce two types of spores that give rise to two types of gametophytes (figure 30.3). The male gametophytes (pollen grains) of pines develop from microspores, which are produced by meiosis in male cones that develop in clusters, typically at the tips of the lower branches; there may be hundreds of such clusters on any single tree. Male cones are typically clustered on the lower branches of pine trees and female cones are often in the upper branches. Pines can produce so much pollen that it can sometimes be seen as a green mist above a pine forest. As the pollen rises from the lower branches of the tree on gentle breezes, it can pollinate the female cones in the upper branches. The male pine cones generally are 1 to 4 cm long and consist of small, papery scales arranged in a spiral or in whorls. A pair of 628 part V Diversity of Life on Earth

microsporangia form as sacs within each scale. Numerous microspore mother cells in the microsporangia undergo meiosis, each becoming four microspores. The microspores divide by mitotic cell division to produce four-celled pollen grains, each with a pair of air sacs that give them added buoyancy when released into the air. A single cluster of male pine cones may produce more than a million pollen grains. Two ovules develop toward the base of each scale. Each ovule contains a megasporangium called the nucellus. The nucellus itself is completely surrounded by a thick layer of cells called the integument that has a small opening (the ­micropyle) toward one end. One of the layers of the integument later becomes the seed coat. A single megaspore mother cell within each megasporangium undergoes meiosis, becoming a row of four megaspores. Three of the megaspores break down, but the remaining one, over the course of about a year, slowly develops into a female gametophyte through mitotic cell divisions. The female gametophyte at maturity may consist of thousands of cells, with two to six archegonia formed at the micropylar end. Each archegonium contains an egg so large it can be seen without a microscope.

Fertilization and seed formation Female cones usually take two or more seasons to mature. At first they may be reddish or purplish in color, but they soon turn green, and during the first spring, the scales spread apart. While the scales are open, pollen grains carried by the wind drift between them, some catching in sticky fluid oozing out of the micropyle. The pollen grains within the sticky fluid are slowly drawn down through the micropyle to the top of the nucellus, and the scales close shortly thereafter. The archegonia and the remainder of the female gametophyte are not mature until about a year later. While the female gametophyte is developing, a pollen tube emerges from a pollen grain at the bottom of the micropyle and slowly digests its way through the nucellus to the archegonia. During growth of the pollen tube, one of the pollen grain’s four cells, the generative cell, divides by mitotic cell division, with one of the resulting two cells dividing once more. These last two cells function as sperm. The germinated pollen grain with its two sperm is the mature male gametophyte, a small and simple haploid structure compared with fern gametophytes. About 15 months after pollination, the pollen tube enters the ovule through the micropyle and discharges its contents. One sperm unites with the egg, forming a zygote. This is the beginning of the sporophyte generation. The other sperm and cells of the pollen grain degenerate. The zygote develops into an embryo surrounded by female gametophyte cells, which provide nutrition. The integument hardens to produce a seed coat. After dispersal and germination of the seed, the young sporophyte of the next generation develops into a tree.

Cycads resemble palms, but are not flowering plants Cycads (phylum Cycadophyta) are slow-growing gymnosperms of tropical and subtropical regions. The sporophytes of most of the 250 known species resemble palm trees (figure 30.4a) with trunks that can attain heights of 15 m or more. Unlike palm trees, which

Pollen

Microspores

Air bladder Pollination

Microspore mother cell

MEI

Pollen tube

OSI

Scale

S

Pollen tube Megaspore mother cell

MI

TO

Sperm

SIS

Megaspore

n 2n

Pollen-bearing cone on lower parts of tree

Seed-bearing cone on upper parts of tree

(15 months after pollination)

FE

RT

ILIZ

AT IO

N

Zygote

Sporophyte MITOSIS

Seedling

MITOSIS

Pine seed

Section of seed (second year), showing embryo embedded in megagametophyte

Figure 30.3 Life cycle of a typical pine. The 2n stage is the sporophyte, whereas the n stage is the gametophyte. Wind generally disperses the male gametophyte (pollen), which carries sperm. Pollen tube growth delivers the sperm to the egg on the female cone. Additional protection for the embryo is provided by the integument, which develops into the seed coat. are flowering plants, cycads produce cones and have a life cycle similar to that of pines. Individuals of the species make either pollen-bearing or ovule-bearing cones, but not both. As such, members of the cycads could be considered to have separate sexes.

The female cones, which develop upright among the leaf bases, are huge in some species and can weigh up to 45 kg. The sperm of cycads, although formed within a pollen tube, are released within the ovule to swim to an archegonium. Several

Figure 30.4 Three phyla of gymnosperms. 

a. A cycad, Cycas circinalis. b. Welwitschia mirabilis represents one of the three genera of gnetophytes. c. Maidenhair tree, Ginkgo biloba, the only living representative of the phylum Ginkgophyta. (a) Luca Invernizzi

Tetto/AGE Fotostock; (b) Juan Carlos Muñoz/AGE Fotostock; (c) Nancy Nehring/Getty Images

a.

b.

c. chapter

30 Seed Plants 629

Only one species of the ginkgophytes remains extant The fossil record indicates that members of the ginkgophytes (phylum Ginkgophyta) were once widely distributed, particularly in the northern hemisphere; today, only one living species, Ginkgo biloba, remains (figure 30.4c). This tree, which sheds its leaves in the fall, was first encountered by Europeans in cultivation in Japan and China; it apparently no longer exists in the wild. Like the sperm of cycads, those of Ginkgo have flagella. The ginkgo is dioecious—that is, the male and female reproductive structures are produced on separate trees. The fleshy outer coverings of the seeds of female ginkgo plants exude the foul smell of rancid butter, caused by the presence of butyric and isobutyric acids, which are also found in butter and vomit. Because of its beauty and resistance to air pollution, Ginkgo is commonly planted along city streets; however, due to the rancid smell produced by female gingko plants, male plants produced by vegetative propagation are preferred for this use. 630 part V Diversity of Life on Earth

■■

What adaptation do conifers exhibit to capture wind-borne pollen?

30.3 Angiosperms:

The Flowering Plants

Learning Outcomes 1. List the defining features of angiosperms. 2. Describe the roles of some animals in the angiosperm life cycle. 3. Explain double fertilization and its outcome.

Angiosperms are a group of seed plants comprising some 300,000 species of flowering plants. Unlike gymnosperms, angiosperms have ovules that are enclosed within diploid tissue at the time of pollination. The ­carpel, a modified leaf that encapsulates seeds, develops into the fruit, a unique angiosperm feature (­figure 30.5). Although some gymnosperms, including the yew (Taxus species), have fleshlike tissue around their seeds, it is of a different origin and not a true fruit. Angiosperms

There are three genera and about 65 living species of g­ netophytes (phylum Gnetophyta). They are the only gymnosperms with vessels in their xylem. Vessels are a particularly efficient conducting cell type that is a common feature in angiosperms. The members of the three genera differ greatly from one another in form. One of the most bizarre of all plants is Welwitschia, which occurs in the Namib and Mossamedes deserts of southwestern Africa (figure 30.4b). The stem is shaped like a large, shallow cup that tapers into a taproot below the surface. It has two strap-shaped, leathery leaves that grow continuously from their base, splitting as they flap in the wind. The reproductive structures of Welwitschia are conelike, appear toward the bases of the leaves around the rims of the stems, and are produced on separate male and female plants. More than half of the gnetophyte species are in the genus Ephedra, which is common in arid regions of the western United States and Mexico. Species are found on every continent except Australia. The plants are shrubby, with stems that superficially resemble those of horsetails, being jointed and having tiny, scalelike leaves at each node. Male and female reproductive structures may be produced on the same or different plants. The drug ephedrine, widely used in the treatment of respiratory problems, was in the past extracted from the Chinese species of Ephedra, but it has now been largely replaced with synthetic preparations (pseudoephedrine). Because ephedrine found in herbal remedies for weight loss was linked to strokes and heart attacks, it was withdrawn from the market in April of 2004. Sales restrictions were placed on ­pseudoephedrine-containing products in 2006 because it can be used to manufacture the illegal drug methamphetamine. The best-known species of the third genus, Gnetum, is a tropical tree, but most species are vinelike. All species have broad leaves similar to those of angiosperms. One Gnetum species is cultivated in Java for its tender shoots, which are cooked as a vegetable.

Gymnosperms are mostly cone-bearing seed plants. In gymnosperms, the ovules are not completely enclosed by sporophyte tissue at pollination, and thus have “naked seeds.” The four groups of gymnosperms are conifers, cycads, gnetophytes, and ginkgophytes.

Gymnosperms

Gnetophytes have xylem vessels

Learning Outcomes Review 30.2

Ferns and Allies

species of cycads are facing extinction in the wild and soon may exist only in botanical gardens.

Sorting out angiosperm origins is complicated The origins of the angiosperms puzzled even Darwin, who referred to their origin as an “abominable mystery.” What particularly puzzled Darwin was the very rapid diversification and success of the angiosperms. Notice in table 30.1 that there are over 300,000 species of angiosperms, but only about 1000 species of all other seed plants combined. The rapid explosion of angiosperm diversity changed Earth’s landscape from one dominated by ferns, cycads, and conifers to the more varied and familiar ecosystems we see today. There are many potential explanations for the takeover of the world’s biomes by the angiosperms. One hypothesis is that the breakup of Pangea (a supercontinent that broke up around 175 mya) led to climate change and opportunities for the expansion of the angiosperms. The middle latitudes became more humid and temperate, and deserts between the tropic and temperate zones became smaller. The ability of angiosperms to move into these new environments was enhanced by their unique features, including flower production, the ability to attract insect pollinators, and the development of broad leaves with dense veins.

Figure 30.5  Evolution of fruit. 

Unlike gymnosperms, the ovule of angiosperms is surrounded by sporophyte tissue derived from leaves. This tissue is called the carpel and develops into the fruit. The leaf origins of carpels are still visible in the pea pod.  Goodshoot/Alamy

Ovules (seeds)

Stock Photo

Carpel (fruit)

Ovules Cross section

Modified leaf with ovules

Folding of leaf protects ovules

Fusion of leaf margins

In Liaoning province of China, a complete angiosperm fossil that is at least 125 million years old has been found (figure 30.6). Archaefructus fossils have both male and female reproductive structures; however, they lack the sepals and petals that evolved in later angiosperms to attract pollinators. The fossils were so well preserved that fossil pollen could be examined using scanning electron microscopy. Although Archaefructus is ancient, it is unFruits

likely to be the very first angiosperm. Still, the incredibly wellpreserved fossils provide valuable detail on angiosperms between the Late Jurassic and the Early Cretaceous periods, when dinosaurs roamed the Earth. Consensus has also been growing on the most basal living angiosperm—Amborella trichopoda (figure 30.7). Amborella, with small, cream-colored flowers, is even more primitive than water lilies. This small shrub, found only on the island of New Caledonia in the South Pacific, is the last remaining species of the earliest extant lineage of the angiosperms that arose about 135 mya. Although Amborella is not the original angiosperm, it is sufficiently close that studying its reproductive biology may help us understand the early radiation of the angiosperms. The angiosperm phylogeny reflects an evolutionary hypothesis that is driving new research on angiosperm origins (figure 30.8).

Paired stamens

carpel

petal

Figure 30.6 Fossil of the oldest known angiosperm. Archaefructus fossil

with multiseeded carpels (fruits) and stamens. This plant is estimated to have lived between 122 and 145 mya.

©David Dilcher and Ge Sun

sepal

Figure 30.7 An ancient living angiosperm, Amborella trichopoda. This plant is believed to be the closest living relative to the original angiosperm. Courtesy of Sandra Floyd

chapter

30 Seed Plants 631

Gymnosperms Ginkgo

Gnetophytes

Conifers

Angiosperms Cycads

Archaefructus (extinct)

Amborella

Water lilies

Star anis

Eudicots

Magnoliids

Monocots

Figure 30.8 Archaefructus may be the sister clade to all other angiosperms. All members of the Archaefructus lineage are extinct, leaving Amborella as the most basal living angiosperm.

Horizontal gene transfer occurred in land plants

Flowers house the gametophyte generation of angiosperms

Amborella trichopoda is the closest living relative to the earliest angiosperms, yet 20 out of 31 of its known mitochondrial protein genes hopped into the mitochondrial genome from other land plants through horizontal gene transfer (HGT). In addition, three different moss species contributed to the mix (figure 30.9).

Flowers are considered to be modified stems bearing modified leaves. Regardless of their size and shape, they all share certain features (figure 30.11). Each flower originates as a ­primordium that develops into a bud at the end of a stalk called a pedicel. The pedicel expands slightly at the tip to form the receptacle, to which the remaining flower parts are attached.

?

Inquiry question Explain why a phylogenetic tree based on comparisons of a single gene could result in an inaccurate hypothesis.

Amborella is not typical of most extant flowering plants. It is the only existing member of its genus and is native only to the tropical rainforests of New Caledonia, an island group east of Australia that has been isolated for some 70 million years and contains many ancient endemic species. Here, parasitic plants (plants that derive nutrients from other plants) are common. Close contact with parasitic plants could increase the probability of HGT (figure 30.10). An open question is whether the moss genes in Amborella have functions. About half the genes are intact and could be expressed to make protein. The protein would be similar to an existing protein in the plant, but its function, if any, remains to be determined.

?

Inquiry question How would you determine if a moss gene in Amborella had a function?

632 part V Diversity of Life on Earth

Flower morphology The other flower parts typically are attached in circles called whorls. The outermost whorl is composed of sepals. Most flowers have three to five sepals, which are green and somewhat leaflike. The next whorl consists of petals that are often colored, attracting pollinators such as insects, birds, and some small mammals. The petals, which also commonly number three to five, may be separate, fused together, or missing altogether, in wind-pollinated flowers. The third whorl consists of stamens and is collectively called the androecium. This whorl is where the male gametophytes, pollen, are produced. Each stamen consists of a pollenbearing anther and a stalk called a filament, which may be missing in some flowers. At the center of the flower is the fourth whorl, the ­gynoecium, where the small female gametophytes are housed; the gynoecium consists of one or more carpels. The first carpel was derived from a leaflike structure with ovules along its margins. Primitive flowers can have several to many separate carpels, but

Moss species 4 - gene C

Amborella - gene C (HGT)

Moss species 3 - gene B

Moss species 2 - gene B

Amborella - gene B (HGT)

Moss species 1 - gene A

Amborella gene A (HGT)

Charales

Mosses

Ferns

Pines

Flowering plants

Mitochondrial Genes A–C

a.

b.

Figure 30.9 The flowering plant Amborella acquired three moss genes through horizontal gene transfer (HGT). 

a. Phylogenetic relationship of Amborella to other land plants. As shown by the arrow connecting moss and the flowering plants, HGT is the only plausible explanation for the presence of moss mitochondrial genes in Amborella. b. Phylogenetic relationships among the horizontally transferred genes.

in most flowers, two to several carpels are fused together. Such fusion can be seen in an orange sliced in half; each segment represents one carpel.

Structure of the carpel

Figure 30.10 Close contact between species can lead to HGT. Here moss is growing on the base of an Amborella leaf with

lichens scattered on the rest of the leaf.  Sarah Mathews, Shirley C. Tucker Chair in Plant Systematics, Department of Biological Sciences, Louisiana State University.

Figure 30.11 Diagram of an angiosperm flower.  a. The main structures of the flower are labeled. b. Details of an ovule. The ovary as it matures will become a fruit; as the ovule’s outer layers (integuments) mature, they will become a seed coat.

A carpel has three major regions (figure 30.11a). The ­ovary is the swollen base, which contains from one to hundreds of ovules; the ovary later develops into a fruit. The tip of the carpel is called a stigma. Most stigmas are sticky or feathery, trapping pollen grains that land on them. Typically, a neck or stalk called a style connects the stigma and the ovary; in some flowers, the style may be very short or even missing. Many flowers have nectar-secreting glands called nectaries, often located toward the base of the ovary. Nectar is a fluid containing sugars, amino acids, and other molecules that attracts insects, birds, and other animals to flowers.

Stigma Carpel

Style Ovule Ovary wall

Petal Sepal

Stamen

Nucellus Megaspore mother cell

Ovary

Integuments

Receptacle Pedicel

a.

Anther Filament

Micropyle Stalk of ovule (funiculus)

b. chapter

30 Seed Plants 633

Double fertilization is a unique feature of the angiosperm life cycle

closest to the micropyle, one cell functions as the egg; the other two cells are called synergids. At the other end, the three cells are now called antipodals; they have no apparent function and eventually break down and disappear. At the same time, two layers of the ovule, the integuments, differentiate and become the seed coat of a seed. The integuments, as they develop, form the micropyle, a small gap or pore at one end that was described in section 30.2 (see figure 30.11b). The large sac with eight nuclei in seven cells is called an embryo sac; it constitutes the female gametophyte. Although it is completely dependent on the sporophyte for nutrition, it is a multicellular, haploid individual.

During development of a flower bud, a single diploid megaspore mother cell in the ovule undergoes meiotic cell division, producing four haploid megaspores (figure 30.12). In most flowering plants, three of the megaspores soon disappear; the nucleus of the remaining megaspore divides mitotically, and the cell slowly expands until it becomes many times its original size.

The female gametophyte While the expansion of the megaspore is occurring, each of the daughter nuclei divides twice, resulting in eight haploid nuclei arranged in two groups of four. One nucleus from each group of four migrates toward the center, where they function as polar nuclei. A cell wall forms around the two polar nuclei to create the central cell. Cell walls also form around the remaining nuclei. In the group

Pollen production While the female gametophyte is developing, a similar but less complex process takes place in the anthers (figure 30.12). Most anthers have patches of tissue (usually four) that eventually become chambers lined with nutritive cells. The tissue in each patch

MITOSIS

Megaspore Generative cell

Polar nuclei

Tube nucleus

M

Egg

EI OS

MITOSIS

IS

Ovule Megaspore mother cell

Pollen tube Sperm

Pollen Formation of pollen tube

Anther Stigma

Anther Microspore mother cells 2n

Ovary

Tube nucleus Style

n

Adult sporophyte with flower

Young sporophyte

Cotyledons AT I

O

N

Polar nuclei

MI

Seed coat

Endosperm

TO

SIS

Embryo Endosperm

Zygote

DOUBLE FERTILIZATION

GE

RM

IN

Seed

Sperm Egg

Figure 30.12 Life cycle of a typical angiosperm. As in pines, external water is no longer required for fertilization. In many species of angiosperms, animals carry pollen to the carpel. The outer wall of the carpel forms the fruit, which often entices animals to eat and, therefore, disperse the seed.

634 part V Diversity of Life on Earth

is composed of many diploid microspore mother cells that undergo meiotic cell division, each producing four microspores. The four microspores at first remain together as a quartet, or tetrad, and the nucleus of each microspore divides once; in most species, the microspores of each quartet then separate. At the same time, a two-layered wall develops around each microspore. As the anther continues to mature, the wall between adjacent pairs of chambers breaks down, leaving two larger sacs. At this point, the binucleate microspores have become pollen grains. The outer pollen grain wall layer often becomes ornately sculptured, and contains chemicals that may react with others in a stigma to signal whether development of the male gametophyte should proceed to completion. The pollen grain has areas called apertures, through which a pollen tube may later emerge.

Pollination and the male gametophyte Pollination is simply the mechanical transfer of pollen from its source (an anther) to a receptive area (the stigma of a flowering plant). Most pollination takes place between flowers of different plants and is brought about by wind, water, gravity, insects, or other animals. In some angiosperms, plants may pollinate themselves in a process called self-pollination. If the stigma is receptive, then the pollen grain’s cytoplasm absorbs substances from the stigma and bulges through a pore in the pollen wall. The bulge develops into a pollen tube that responds to chemical and mechanical stimuli that guide it through the style and into the micropyle (opening) of the ovule to the embryo sac. The pollen tube usually takes several hours to two days to reach the

micropyle, but in a few instances, the journey may take up to a year. Pollen tube growth is more rapid in angiosperms than in gymnosperms. Rapid pollen tube growth rate is an innovation that is believed to have preceded fruit development and been essential in the origins of angiosperms (figure 30.13). The nucleus of one of the pollen grain’s two cells, the generative cell, divides in the pollen grain or in the pollen tube, producing two sperm cells. Unlike sperm in mosses, ferns, and some gymnosperms, the sperm of flowering plants have no flagella.

Double fertilization and seed production As the pollen tube enters the embryo sac, it destroys a synergid and then discharges its contents. Both sperm are functional, and an event called double fertilization follows. One sperm unites with the egg and forms a zygote, which develops into an embryo sporophyte plant. The other sperm and the two polar nuclei unite, forming a triploid primary endosperm nucleus. Double fertilization is unique to the angiosperms. Consequently, only the flowering plants produce endosperm. The evolutionary advantage of double fertilization remains unclear. Some suggest that by delaying the production of nutritive tissue until the point of fertilization, the plant saves precious resources. Others have suggested that the rapid divisions of the endosperm could be an advantage. Rapid seed formation likely reduces the probability that the seed will be consumed. The primary endosperm nucleus begins dividing rapidly and repeatedly, becoming triploid endosperm tissue that may soon consist of thousands of cells. Endosperm tissue can become an extensive part of the seed in grasses such as corn, and it provides

SCIENTIFIC THINKING Hypothesis: An increase in pollen tube growth rate accompanied angiosperm origins. Prediction: Pollen tube growth rates will be slower in gymnosperms than in close relatives of the first angiosperm. Test: Pollinate three angiosperm species, including Amborella, that are most closely related to the first angiosperm. At different time intervals, remove the style and measure the lengths of pollen tubes using a fluorescence microscope. Calculate and compare the rate of pollen tube growth with results published for gymnosperms.

Close relatives of ancestral angiosperm Amborella trichopoda Nuphar polysepala Austrobaileya scandens Gymnosperms Gnetum gnemon Ginkgo biloba

Pollen Tube Growth Rate (μm/hour)

80.0 589.2 271.7

6.4 0.3

Pollen Tube Growth Rate of Amborella trichopoda

Length of pollen tube (μm)

Species

640 560

Style

480 400

Pollen tubes

320 240 160 80 0

Ovule 0

1

2 3 4 5 6 7 Hours after pollination

8

Conclusion: Pollen tube growth rate is higher in relatives of ancestral angiosperms than in gymnosperms, supporting the hypothesis. Further Experiments: Develop a hypothesis to explain the difference in pollen tube growth rates among the tested angiosperms. Is there a correlation between the distance a pollen tube must travel to fertilize an egg and the rate of growth? How could you test your hypothesis?

Figure 30.13 Rapid pollen tube growth accompanied the origin of angiosperms. Dr. Joseph Williams chapter

30 Seed Plants 635

nutrients for the embryo in most flowering plants (see figure 40.27). The endosperm nourishes the embryo in angiosperms, whereas the female gametophyte serves that function in gymnosperms.

?

Inquiry question If the endosperm failed to develop in a seed, how do you think the fitness of that seed’s embryo would be affected? Explain your answer.

Shoot apical meristem

Data analysis Using the graph in figure 30.13, calculate the pollen tube growth rate, being sure to show your work. Compare your rate with the rate in the table in figure 30.13 and propose an explanation for any differences.

Seed coat (integuments)

Learning Outcomes Review 30.3

Amborella, the closest living relative of the first angiosperms, provides clues to the origins of this very successful group. Although angiosperms are a clade, horizontal gene transfer has contributed genes from distantly related plant species. Angiosperms are characterized by ovules that at pollination are enclosed within an ovary at the base of a carpel, a structure unique to the phylum; the ovary develops into a fruit. Evolutionary innovations of angiosperms include flowers to attract pollinators, fruits to protect embryos and aid in their dispersal, rapid pollen tube growth, and double fertilization, which provides endosperm to help nourish the embryo. ■■

How could genes from moss be transported into a flowering plant, other than via parasitic plants?

30.4 Seeds Learning Outcomes 1. Describe four ways in which seeds help to ensure the survival of a plant’s offspring. 2. List environmental conditions that can lead to seed germination in some plants.

Following double fertilization in angiosperms and single fertilization in gymnosperms that produces an embryo, a profoundly important event occurs: the embryo stops developing. In this section, we focus on seed development in the angiosperms. In many plants, development of the embryo is arrested soon after the meristems and the cotyledons (seed leaves) form. The integuments— the outer cell layers of the ovule—develop into a relatively impermeable seed coat, which encloses the seed, which contains a dormant embryo and stored food (figure 30.14).

Seeds protect the embryo The seed is a vehicle for dispersing the embryo to distant sites. Being encased in the protective layers of a seed allows a plant embryo to survive in environments that might kill a mature plant. 636 part V Diversity of Life on Earth

Procambium Root apical meristem Root cap Endosperm

Cotyledons

Figure 30.14 Seed development. The integuments of this mature angiosperm ovule are forming the seed coat. Note that the two cotyledons have grown into a bent shape to accommodate the tight confines of the seed. Seeds are an important adaptation in at least four ways: 1. Seeds maintain dormancy under unfavorable conditions and postpone development until better conditions arise. 2. Seeds afford maximum protection to the young plant at its most vulnerable stage of development. 3. Seeds contain stored food that allows a young plant to grow and develop before photosynthetic activity begins. 4. Perhaps most important, seeds are adapted for dispersal, facilitating the migration of plant genotypes into new habitats. A mature seed contains only about 5 to 20% water. Under these conditions, the seed and the embryonic plant within it are very stable; the embryo’s arrested growth is primarily due to progressive and severe desiccation and the associated reduction in metabolic activity. Germination cannot take place until water and oxygen reach the embryo. Seeds of some plants have been known to remain viable for hundreds and, in rare instances, thousands of years.

Specialized seed adaptations improve survival Specific adaptations often help ensure that seeds will germinate only under appropriate conditions. Sometimes, seeds lie within tough cones that do not open until they are exposed to the heat of a fire (figure 30.15). This strategy causes the seed to germinate in an open, fire-cleared habitat where nutrients are relatively abundant, having been released from plants burned in the fire.

30.5 Fruits Learning Outcomes 1. Identify the structures from which fruits develop. 2. Distinguish among berries, legumes, drupes, and samaras.

aa.

Survival of angiosperm embryos depends on fruit development as well as seed development. Fruits are most simply defined as mature ovaries (carpels). During seed formation, the ovary begins to develop into fruit (figure 30.16). It is possible for fruits to develop without seeds. Commercial bananas, for example, are fruits that lack viable seeds. Bananas must be propagated asexually.

Fruits are adapted for dispersal Fruits form in many ways and exhibit a wide array of adaptations for dispersal. Three layers of ovary wall, also called the pericarp, can have distinct fates, which account for the ­diversity of fruit Stigma

Style

b.

Figure 30.15 Fire induces seed release in some pines. 

Pericarp (ovary wall) Exocarp Mesocarp Endocarp

Fire can destroy adult jack pines, but stimulate growth of the next generation. a. The cones of a jack pine are tightly sealed and cannot release the seeds protected by the scales. b. High temperatures lead to the release of the seeds. (a) Ed Reschke; (b) NPS Photo by Don Despain

Ovary

Seeds of other plants germinate only when inhibitory chemicals leach from their seed coats, thus guaranteeing their germination when sufficient water is available. Still other seeds germinate only after they pass through the intestines of birds or mammals or are regurgitated by them, which both weakens the seed coats and ensures dispersal. Sometimes seeds of plants thought to be extinct in a particular area may germinate under unique or improved environmental circumstances, and the plants may then reestablish themselves.

Learning Outcomes Review 30.4

The seed coat originates from the integuments and encloses the embryo and stored nutrients. The four advantages conferred by seeds are dormancy, protection of the embryo, nourishment, and a method of dispersal. Fire, heavy rains, or passage through an animal’s digestive tract may be required for germination in some species. ■■

What type of seed dormancy would you expect to find in trees living in climates with cold winters?

Part of ovary developing into seed

Developing seed coat Embryo Endosperm

prior sporophyte generation degenerating gametophyte generation next sporophyte generation

Carpel (developing fruit)

Figure 30.16 Fruit development. The carpel (ovary) wall is composed of three layers: the exocarp, mesocarp, and endocarp. One, some, or all of these layers develop to contribute to the recognized fruit in different species. The seed matures within this developing fruit.

?

Inquiry question Three generations are represented in this diagram. Label the ploidy levels of the tissues of different generations shown here.

chapter

30 Seed Plants 637

True Berries

Drupes

Outer pericarp The entire pericarp is Fused Seed fleshy, with a thin skin. carpels Berries have multiple seeds in one or more ovaries. The tomato flower that produced this berry had four carpels that fused. Each carpel contains multiple ovules, each of which develops into a seed.

Aggregate Fruits

Pericarp A single seed Exocarp (skin) is enclosed Mesocarp (flesh) in a hard pit; Endocarp (pit) peaches, plums, cherries. Each layer of the pericarp has a different structure and function, with Seed the endocarp

Derived from many Sepals of a ovaries of a single single flower flower; strawberries, blackberries. Unlike tomato, Ovary these ovaries Seed are not fused.

forming the pit.

Samaras

Legumes

Multiple Fruits

The fruit splits along two carpel edges (sutures) with seeds attached to edges; peas, beans. Unlike in fleshy fruits, the three tissue layers of the ovary wall do not thicken extensively. The entire pericarp is dry at maturity. Pericarp

Stigma

Seed

Style

The fruit does not split. A wing forms from the outer tissues; maples, elms, ashes.

Pericarp

Seed

The individual flowers form fruits around a single stem. The fruits fuse as seen with pineapple.

Main stem Pericarp of individual flower

Figure 30.17 Examples of some kinds of fruits. Legumes and samaras are examples of dry fruits. Legumes open to release their seeds,

but samaras do not. Drupes and true berries are simple fleshy fruits; they develop from a flower with a single pistil composed of one or more carpels. Aggregate and multiple fruits are compound fleshy fruits; they develop from flowers with more than one pistil or from more than one flower.  (top left)

Kingsley Stern; (top middle) Kingsley Stern; (top right) ©Steven P. Lynch; (bottom left) Ruth Swan/Shutterstock; (bottom middle) Kingsley R. Stern; (bottom right) Charles D. Winters/ Science Source

types, from fleshy and soft to dry and hard. The differences among some of the fruit types are shown in figure 30.17. Fruits contain three genotypes in one structure. The fruit and seed coat are from the prior sporophyte generation. The embryo represents the next sporophyte generation (see figure 30.16). Finally, the endosperm is a transient, triploid product of fertilization.

Fruits allow angiosperms to colonize large areas The diversity of fruit types contributes to an array of dispersal methods. Fruits with fleshy coverings are normally dispersed by birds or other vertebrates (figure 30.18a). Like red flowers, red fruits signal an abundant food supply. By feeding on these fruits, birds and other 638 part V Diversity of Life on Earth

animals may carry seeds from place to place and thus transfer plants from one suitable habitat to another. Such seeds require a hard seed coat to resist stomach acids and digestive enzymes. Fruits with hooked spines, such as those of burrs (­figure 30.18b), are typical of several genera of plants that occur in the northern deciduous forests. Such fruits are often disseminated by mammals, including humans, when they hitch a ride on fur or clothing. Squirrels and similar mammals disperse and bury fruits such as acorns and other nuts. Some of these sprout when conditions become favorable, such as after the spring thaw. Other fruits, including those of maples, elms, and ashes, have wings that aid in their distribution by the wind. Orchids have minute, dustlike seeds, which are likewise blown away by the wind. The dandelion provides another familiar example of a fruit

aa.

bb.

cc.

dd.

Figure 30.18 Mechanisms for fruit dispersal. a. The bright red berries of this honeysuckle, Lonicera hispidula, are highly attractive to birds. After eating the fruits, birds may carry the seeds they contain for great distances either internally or, because of their sticky pulp, stuck to their feet or other body parts. b. You will know if you have ever stepped on the fruits of Cenchrus incertus; their spines adhere readily to any passing animal. c. False dandelion, Pyrrhopappus carolinianus, has “parachutes” that widely disperse the fruits in the wind, much to the gardener’s despair. d. This fruit of the coconut palm, Cocos nucifera, is sprouting on a sandy beach. Coconuts, one of the most useful fruits for humans in the tropics, have become established on other islands by drifting there on waves.  (a) BeppeNob/Shutterstock; (b) ©Steven P. Lynch; (c) Brian Jackson/123RF; (d) John Kaprielian/Science Source

type that is wind-dispersed (figure 30.18c), and the dispersal of seeds from plants such as milkweeds, willows, and cottonwoods is similar. Water dispersal adaptations include air-filled chambers surrounded by impermeable membranes to prevent waterlogging. Coconuts and other plants that characteristically occur on or near beaches are regularly spread throughout a region by floating in water (figure 30.18d). This sort of dispersal is especially important in the colonization of distant island groups, such as the Hawaiian Islands. It has been calculated that the seeds of about 175 angiosperms, nearly one-third from North America, must have reached Hawaii to evolve into the roughly 970 species found there today. Some of these seeds blew through the air, others were transported on the feathers or in the guts of birds, and still others floated across the Pacific. Although the distances are rarely as great as the

distance between Hawaii and the continental United States, dispersal is just as important for mainland plant species that have discontinuous habitats, such as mountaintops, marshes, or rivers.

Learning Outcomes Review 30.5

As a seed develops, the pericarp layers of the ovary wall develop into the fruit. A berry has a fleshy pericarp; a legume has a dry pericarp that opens to release seeds; the outer layers of a drupe pericarp are fleshy; and a samara is a dry structure with a wing. Animals often distribute the seeds of fleshy fruits and fruits with spines or hooks. Wind disperses lightweight seeds and samaras. ■■

What features of fruits might encourage animals to eat them?

Chapter Review Zoonar/O Popova/age fotostock

30.1 The Evolution of Seed Plants

30.2 Gymnosperms: Plants with “Naked Seeds”

The seed protects the embryo. An extra layer of sporophyte tissue surrounds the embryo, creating the ovule, which later hardens (figure 30.1). The seed protects the embryo, helps it resist drying out, and allows a dormant stage that pauses the life cycle until environmental conditions are favorable.

Gymnosperms have ovules that are not completely enclosed in sporophyte (diploid) tissue at the time of pollination. The four groups of gymnosperms are coniferophytes, cycadophytes, gnetophytes, and ginkgophytes; all lack flowers and true fruits.

Water is not required to transport the male gametophyte to the female gametophyte. The gametophytes of seed plants typically consist of only a few cells. Pollen grains are male gametophytes; each pollen grain contains sperm cells. Water is not required for fertilization. The female gametophyte develops within an ovule that forms the seed.

(figure 30.3)

Conifers are the largest gymnosperm phylum. Conifers include pines, spruces, firs, cedars, and many other groups. Both the tallest and the oldest vascular plants are conifers. Pines are an exemplary conifer genus. Pines have tough, needle-like leaves in clusters of two to five. They produce male and female cones. chapter

30 Seed Plants 639

In the male cones, microspore mother cells in the microsporangia give rise to microspores that then develop into four-celled pollen grains, the male gametophytes. In the female cones, a megasporangium (nucellus) produces a single megaspore mother cell that becomes four megaspores; three of these break down, and the remaining one develops into a female gametophyte, which produces archegonia that carry eggs. Upon fertilization, a pollen tube emerges from the pollen grain and grows through the nucellus. Eventually two sperm cells migrate through the tube, and one unites with the egg; the other disintegrates. Cycads resemble palms, but are not flowering plants. Most cycads can reach heights of 15 m or more. Cycads, unlike palms, produce cones. Their life cycle is like that of conifers. Gnetophytes have xylem vessels. Xylem vessels, a common feature in angiosperms, are highly efficient conducting cells. Gnetophytes are the only gymnosperms that have them. This group includes the strange Welwitschia genus of southwestern Africa and the numerous worldwide Ephedra species. Only one species of the ginkgophytes remains extant. Ginkgo biloba is a gymnosperm with broad leaves that it sheds in the fall. It is dioecious, and the fleshy seeds of the female tree have a foul odor.

30.3 Angiosperms: The Flowering Plants (figure 30.8) Angiosperms are distinct from gymnosperms and other plants because their ovules are enclosed within diploid tissue called the ovary at the time of fertilization, and they form fruits. Sorting out angiosperm origins is complicated. No one is certain how the angiosperms arose, although the breakup of Pangea may have led to climate change that favored the expansion of the angiosperms. The earliest extant angiosperm appears to be Amborella trichopoda, found on the island of New Caledonia. Horizontal gene transfer occurred in land plants. Moss genes have been found in the Amborella genome, possibly transferred by parasitic plants, although it is unknown if they have a function in Amborella. Flowers house the gametophyte generation of angiosperms. Flowers are considered to be modified stems that bear modified leaves. Flower parts are organized into four whorls: sepals, petals, androecium, and gynoecium (figure 30.11a). The male androecium consists of the stamens, where haploid pollen, the male gametophyte, is produced.

640 part V Diversity of Life on Earth

The female gynoecium consists of one or more carpels that contain the female gametophyte. The carpel has three major regions: the ovary, which later becomes the fruit; the stigma, which is the tip of the carpel; and the style, a stalk that connects the stigma and the ovary. Double fertilization is a unique feature of the angiosperm life cycle (figure 30.12). The megaspore produces eight haploid nuclei. The female gametophyte consists of a large embryo sac with the eight nuclei in seven cells. The egg and the two polar nuclei (in a single cell) are most important. After landing on a receptive stigma, a pollen grain develops a pollen tube that grows toward the embryo sac. Eventually, two sperm pass through this tube. One fuses with the egg to form a zygote, and the other unites with the polar bodies to form a triploid endosperm nucleus that develops into endosperm to nourish the embryo.

30.4 Seeds (figure 30.14) Seeds protect the embryo. Seeds help to ensure the survival of the next generation by maintaining dormancy during unfavorable conditions, protecting the embryo, providing food for the embryo, and providing a means for dispersal. Specialized seed adaptations improve survival. Before a seed germinates, its seed coat must become permeable so that water and oxygen can reach the embryo. Adaptations have evolved to ensure germination under appropriate survival conditions. In certain gymnosperms, seeds may be released from cones after a fire. Alternatively, seeds may require passage through a digestive tract, freeze–thaw cycles, or abundant moisture.

30.5 Fruits (figure 30.16) Fruits are adapted for dispersal. In angiosperms, a fruit is a mature ovary. Fruit development is coordinated with embryo, endosperm, and seed coat development. Angiosperms produce many types of fruit, which vary depending on the fate of the pericarp (carpel wall). Fruits can be dry or fleshy, and they can be simple (single carpel), aggregate (multiple carpels), or multiple (multiple flowers). A fruit is genetically unique because it contains tissues from the parent sporophyte (the seed coat and fruit tissue), the gametophyte (remnants in the developing seed), and the offspring sporophyte (the embryo). Fruits allow angiosperms to colonize large areas. Fruits exhibit a wide array of dispersal mechanisms. They may be ingested and transported by animals, be buried in caches by herbivores, be carried away by birds and mammals, be blown by the wind, or float away on water.

Visual Summary do not require

Water for fertilization

provide protection for the

Seed Plants

Embryo

include make Partially enclosed seeds

produce

Gymnosperms

Flowers

many Coniferophytes produce

Cycadophytes

include Cones

Spruces

contain

Gnetophytes

Ginkgophytes

produce

release

Enclosed seeds

Pollen

within Zygote

contained within

produce

Cones

Pines

Carpels

Anthers classified as

Firs

have

Angiosperms

only resemble gymnosperms with

Cedars

Palms

Vessels in xylem

Many others

include

produces

Fruits

Ovule within ovary

contains

contains

Sperm

Egg

Double fertilization Ginkgo biloba

include

True berries

Aggregate fruit

Samaras

Drupes

Legumes

Multiple fruits

Review Questions Zoonar/O Popova/age fotostock

U N D E R S TA N D

1. The lack of seeds is a characteristic of all a. lycophytes. b. conifers.

c. tracheophytes. d. gnetophytes.

2. Which of the following adaptations allows plants to pause their life cycle until environmental conditions are optimal? a. Stomata b. Phloem and xylem

c. Seeds d. Flowers

3. Which of the following gymnosperms possesses a form of vascular tissue that is similar to that found in the angiosperms? a. Cycads b. Gnetophytes

c. Ginkgophytes d. Conifers

4. In a pine tree, the microspores and megaspores are produced by the process of a. fertilization. b. mitotic cell division.

c. fusion. d. meiotic cell division.

5. Which of the following terms is NOT associated with a male part of a plant? a. Megaspore c. Pollen grains b. Antheridium d. Microspore 6. Which of the following potentially represents the oldest known living species of angiosperm? a. Cooksonia c. Chlamydomonas b. Archaefructus d. Amborella 7. The integuments of an ovule will develop into the a. embryo. b. endosperm. 8. An example of a drupe is a

c. fruit. d. seed coat.

a. strawberry. b. plum. 9. The pericarp is the

c. bean. d. pineapple.

a. ovary wall. b. developing seed coat.

c. ovary. d. mature endosperm. chapter

30 Seed Plants 641

A P P LY 1. Reproduction in angiosperms can occur more quickly than in gymnosperms because a. gymnosperm sperm requires water to swim to the egg. b. flowers always increase the rate of reproduction. c. angiosperm pollen tubes grow more quickly than gymnosperm pollen tubes. d. angiosperms have nectaries. 2. In double fertilization, one sperm produces a diploid ___, and the other produces a triploid ___. a. b. c. d.

zygote; primary endosperm primary endosperm; microspore antipodal; zygote polar nuclei; zygote

3. Which of the following innovations likely contributed to the tremendous success of angiosperms? a. b. c. d.

Homospory in angiosperms Fruits that attract animal dispersers Cones that protect the seed Dominant gametophyte generation

4. Comparing stems of two plant specimens under the microscope, you identify vessels in only one specimen and conclude the specimen a. b. c. d.

with vessels must be an angiosperm or a gnetophyte. with vessels is either Ephedra or a cycad. without vessels is a pterophyte. without vessels must be a tracheophyte.

5. In a flower after fertilization, the following tissues are diploid: a. carpel, integuments, and megaspore mother cell. b. carpel, integuments, and megaspore.

642 part V Diversity of Life on Earth

c. carpel, megaspore, and zygote. d. carpel, megaspore mother cell, and endosperm. 6. Fruits are complex organs that are specialized for dispersal of seeds. Which of the following plant tissues does NOT contribute to mature fruit? a. b. c. d.

Sporophytic tissue from the previous generation Gametophytic tissue from the previous generation Sporophytic tissue from the next generation Gametophytic tissue from the next generation

SYNTHESIZE 1. You have been hired as a research assistant to investigate the origins of the angiosperms—specifically, the relationship(s) between gymnosperms and angiosperms. Which characteristics would you use to clearly define a new fossil as a gymnosperm? As an angiosperm? 2. Describe the benefits and drawbacks of self-pollination for a flowering plant. 3. The relationship between flowering plants and pollinators is often used as an example of coevolution. Many flowering plant species have flower structures that are adaptive to a single species of pollinator. Evaluate the benefits and drawbacks of using such a specialized relationship. 4. As you are eating an apple one day, you decide that you’d like to save the seeds and plant them. You do so, but they fail to germinate. Discuss the possible reasons that the seeds did not germinate and strategies you could try to improve your chances of success.

31 CHAPTER

Fungi Chapter Contents 31.1

Classification of Fungi

31.2

Fungal Forms, Nutrition, and Reproduction

31.3

Fungal Ecology

31.4

Fungal Parasites and Pathogens

31.5

Basidiomycota: The Club (Basidium) Fungi

31.6

Ascomycota: The Sac (Ascus) Fungi

31.7

Glomeromycota: Asexual Plant Symbionts

31.8

Zygomycota: Zygote-Producing Fungi

31.9

Chytridiomycota and Relatives: Fungi with Zoospores

31.10 Microsporidia: Unicellular Parasites

David Clapp/Getty Images

Introduction

Visual Outline differ in

impact

Fungi

Ecosystems

can be Forms

classified as

Nutritional strategies

Parasitic or pathogenic

Reproduction

Basidiomycota

Glomeromycota

Ascomycota

Chytridiomycota

Zygomycota

Microsporidia

Fungi are found everywhere on Earth— from the tropics to the tundra and in both terrestrial and aquatic environments—and have a profound influence on ecology and human health. Although they are often thought to be closely related to plants, fungi share a more recent common ancestor with animals and are therefore more closely related to animals than they are to plants. This can cause significant problems for the treatment of fungal pathogens of animals: the two groups’ physiological similarities mean that drugs that can kill fungal pathogens may also harm host animals, including humans. Fungi secrete digestive enzymes and then absorb the externally digested nutrients that are rapidly distributed throughout the organism. This allows for rapid growth and adaptation to a wide range of ecological niches. Fungi made it possible for plants to colonize land by associating with rootless stems of plants to aid in the

uptake of nutrients and water. Although some fungi cause disease in plants, they play critical roles in the carbon cycle, breaking down dead and dying plant matter to rapidly mobilize plant-fixed carbon into forms of carbon usable by other organisms. Some fungi, like the fly agaric (Amanita muscaria) pictured here, produce toxins, but other fungi are used as foods by humans, and yeasts have been used for millennia to make breads and alcoholic drinks.

31.1

ancestor was unicellular, and from that point, unique solutions to multicellularity evolved in both fungi and animals. Attempts to resolve the phylogenetic relationships between fungal groups have stimulated much debate. The existing molecular data can be used to create classifications based on alternative hypotheses. Traditionally, four fungal phyla were recognized, ­ based p­ rimarily on characteristics of the cells undergoing meiosis: Chytridiomycota (“chytrids”), Zygomycota (“zygomycetes”), Ascomycota (“ascomycetes”), and Basidiomycota (“basidiomycetes”). The discovery that the Zygomycota and Chytridiomycota were paraphyletic led to the subdivision of the “chytrids” into three distinct phyla: Chytridiomycota, Blastocladiomycota, and Neocallimastigomycota.

Classification of Fungi

Kingdom Fungi contains six monophyletic groups

Learning Outcomes 1. List the phyla included in Kingdom Fungi. 2. Describe how approaches to classifying fungi have changed over time.

There are now six widely recognized monophyletic groups that make up Kingdom Fungi: Blastocladiomycota, Neocallimastigomycota, Chytridiomycota, Glomeromycota, Basidiomycota, and Ascomycota (figure 31.1 and table 31.1). Although the relationships within the paraphyletic Zygomycota have yet to be resolved, most classifications of Kingdom Fungi also include this group. Another group, the Microsporidia, are sometimes included in the kingdom due to their similarities with some of the other groups and a shared ancestor with the Zygomycota; however, they are often not considered “true” fungi despite being studied by mycologists. A relatively new group, the Cryptomycota, has been suggested as a seventh phylum. This group includes a diverse, yet uncultured, group of fungi so far only detected by molecular techniques in soil, and in marine and aquatic environments. Confounding our understanding of the complex evolutionary relationships between fungi is a growing body of evidence that horizontal gene transfer has occurred between many fungal species.

Early attempts at classifying fungi relied on comparisons using their gross morphological features. Advances in microscopy and biochemistry in the 18th, 19th, and 20th centuries allowed refinement of earlier classifications. Then, rapid advances in molecular biology in the 1980s allowed more accurate classifications that better reflected the evolutionary relationships between different groups of fungi. Modern molecular techniques, including genomics, are revealing the unique evolutionary journey that has shaped this kingdom and will continue to inform new classifications and approaches to systematics. Phylogenetic analyses of DNA and protein sequences show that fungi are more closely related to animals than to plants. Fossils and molecular data indicate that animals and fungi last shared a common ancestor at least 460 mya. This

Blastocladiomycota

Zygomycota

Neocallimastigomycota

Chytridiomycota

Glomeromycota

Basidiomycota

Ascomycota

a.

b.

c.

d.

e.

f.

g.

500 μm

300 μm

300 μm

200 μm

300 μm

Ancestor of fungi

Figure 31.1 The major phyla of fungi. All phyla, except Zygomycota, are monophyletic. Not shown here but included in some classifications are Microsporidia. a. Allomyces arbuscula, a water mold, is a blastocladiomycete. b. Pilobolus, a zygomycete, grows on animal dung and on culture medium. c. Neocallimastigomycota, including Piromyces communis, decompose cellulose in the rumens of herbivores. d. Some chytrids, including members of the genus Rhizophydium, parasitize green algae. e. Spores of Glomus intraradices, a glomeromycete associated with roots. f. Amanita muscaria, the fly agaric, is a toxic basidiomycete. g. A cup fungus of the genus Peziza, an ascomycete that grows on the ground, rotting wood, or dung. (a) Contributed by Don Barr, Mycological Society of America; (b) ©McGraw Hill/Richard Gross, photographer; (c) Contributed by Don Barr, Mycological Society of America; (d) Dr. Yuuji Tsukii; (e) ©Yves Prin; (f) inga spence/Alamy Stock Photo; (g) Ihor Hvozdetskyi/Shutterstock

644 part V Diversity of Life on Earth

TA B L E 3 1 .1

Fungi

Group

Typical Examples

Key Characteristics

Approximate Number of Living Species

Chytridiomycota

Batrachochytrium

Aquatic, flagellated fungi that produce haploid gametes by sexual reproduction or diploid zoospores by asexual reproduction.

1000

Zygomycota

Rhizopus, Pilobolus

Multinucleate hyphae lack septa, except for reproductive structures; fusion of hyphae leads directly to formation of a zygote in zygosporangium, in which meiosis occurs just before it germinates; asexual reproduction is most common.

1050

Glomeromycota

Glomus

Form arbuscular mycorrhizae. Multinucleate hyphae lack septa. Reproduce asexually.

150

Ascomycota

Truffles, morels

In sexual reproduction, ascospores are formed inside a sac called an ascus; asexual reproduction is also common.

>30,000

Basidiomycota

Mushrooms, toadstools, rusts

In sexual reproduction, basidiospores are borne on club-shaped structures called basidia; asexual reproduction occurs occasionally.

>30,000

Neocallimastigomycota

Neocallimastix

Fungi lacking mitochondria that use anaerobic metabolism to grow in the guts of herbivores. Possess the ability to degrade cellulose.

20

Blastocladiomycota

Allomyces

Exhibit alternation of generations as seen in plant reproduction. Possess characteristic nuclear cap composed of membrane-bound ribosomes.

Learning Outcomes Review 31.1

Advances in microscopy, biochemistry, and molecular biology have provided some clarification of the evolutionary relationships between different groups of fungi. Physiological and molecular evidence shows that fungi are more closely related to animals than they are to plants. Molecular analyses have resolved Kingdom Fungi into six monophyletic taxa plus the paraphyletic Zygomycota. Microsporidia are studied by mycologists; however, they are not considered true fungi. The Cryptomycota are potentially a new group to be considered within Kingdom Fungi. ■■ ■■

In respect to phylogenetics, how are the Zygomycota different from other taxa of fungi? If a new species of fungus were discovered, what might be the best way to classify it?

31.2

Fungal Forms, Nutrition, and Reproduction

Learning Outcomes 1. Distinguish fungi from other eukaryotes. 2. Compare cell division in fungi and higher eukaryotes. 3. Describe fungal structures involved in vegetative and reproductive growth. 4. Relate the structure of fungi to their nutritional strategy.

We usually become aware of fungi when we see them growing in our lawns, on trees in our yards, or on the bread left out on a counter too long. However, mycologists—scientists who study

c. In this expression, b and c are the benefits and costs of the altruistic act, respectively, and r is the coefficient of relatedness, the proportion of alleles shared by two individuals through common descent. For ­example, an individual should be willing to have one less child (c = 1) if such actions allow a halfsibling, which shares one-quarter of its genes (r = 0.25), to have five or more additional offspring (b = 5).

Unrelated female

Unrelated female FF

AD

TT

WY

77

13

RH

KM

Brothers

AC

EE

XZ

SS

13

88

JM

NQ

DF

EA

WT

SX First Cousins

37

83

KR

NM

Average Genetic Relatedness Parent-offspring Full siblings Half-siblings 1st cousins

= = = =

1/2 1/2 1/4 1/8

Haplodiploidy and altruism in ants, bees, and wasps The relationship between genetic relatedness, kin selection, and ­altruism can be best understood using social insects as an example. A hive of honeybees consists of a single queen, who is the sole ­egg-layer, and tens of thousands of her offspring, female workers with nonfunctional ovaries (figure 53.31). Honey­bees are eusocial (“truly” social): their societies are defined by reproductive division of labor (only the queen reproduces), cooperative care of the brood (workers nurse, clean, and forage), and overlap of generations (the queen lives with several generations of her offspring). Males only have one role: they leave the hive and try to find a queen to mate with and start a new colony. Darwin was perplexed by eusociality. How could natural selection favor the evolution of sterile workers that left no offspring? It remained for Hamilton to explain the origin of eusociality in hymenopterans (bees, wasps, and ants) using his kin selection model. In these insects, males are haploid (produced from unfertilized eggs) and females are diploid. This system of sex determination, called haplodiploidy (see figure 51.2), leads to unusual genetic relatedness among colony members. If the queen is fertilized by a single male, then all female offspring will inherit exactly the same alleles from their father (because he is haploid and has only one copy of each allele). Female offspring (workers and future queens) will also share among themselves, on average, half of the alleles they get from their

Figure 53.30 Hypothetical example of genetic relationships. On average, full siblings share half of their alleles (for illustrative purposes, the parents are shown as each having two different alleles for each gene; for example, the father has alleles A and B for Gene 1, whereas the mother has alleles C and D). By contrast, cousins only share one-eighth of their alleles on average. Each letter and number represents a different allele.

Data analysis On average, how genetically similar are you to your aunt?

Hamilton then defined reproductive success with a new concept: inclusive fitness. Inclusive fitness considers gene propagation through both direct (personal fitness) and indirect (the fitness of relatives) reproduction. Hamilton’s kin selection model predicts that altruism is likely to be directed toward close relatives. 1208 part VIII Ecology and Behavior

Figure 53.31 Reproductive division of labor in honeybees. The queen (center) is the sole egg-layer. Her daughters

are sterile workers. Steve Hopkin/Getty Images

mother, the queen. Consequently, they will share, on average, 75% of their alleles with each sister (to verify this, rework figure 53.30, but allow the father to only have one allele for each gene). Now recall Haldane’s statement of commitment to family while you read this section. If a worker should have offspring of her own, she would share only half of her alleles with her female offspring (the other half would come from their father) in contrast to the three-quarters sharing of alleles with her sisters. Thus, because of the close genetic relatedness of sisters due to haplodiploidy, workers would propagate more of their own alleles by giving up their own reproduction to assist their mother in rearing their sisters, some of whom will be new queens, start new colonies, and reproduce. In this way, the unusual haplodiploid system may have set the “genetic stage” for the evolution of eusociality. Indeed, eusociality has evolved at least 12 separate times in the Hymenoptera. One wrinkle in this theory, however, is that eusocial systems have evolved in other insects (thrips, some weevils, and termites), and mammals (naked mole rats). Although the thrips and weevils are also haplodiploid, termites and naked mole rats are not. Thus, although ­haplodiploidy may have facilitated the evolution of eusociality, other factors can influence social evolution.

Data analysis Suppose that males are haploid (have

only one chromosome, and thus only one allele for each gene), as in hymenopterans. How closely related would female first cousins be if their mothers were sisters? How closely related would they be if, instead, their fathers were brothers (remember that first cousins are the offspring of siblings (either two brothers, two sisters, or a brother and a sister)? You may want to refer to figure 53.30 to help you organize your approach to this question.

Other examples of kin selection Kin selection may explain altruism in other animals. Belding’s ground squirrels (Spermophilus beldingi) give alarm calls when they spot a predator such as a coyote or a badger. Such predators may attack a calling squirrel, so giving the signal places the caller at risk. A ground squirrel colony consists of a female and her daughters, sisters, aunts, and nieces. When males mature, they disperse long distances from where they are born, so adult males in the colony are not genetically related to the females. By marking all squirrels in a colony with an individual dye pattern on their fur and by recording which individuals gave calls and the social circumstances of their calling, researchers found that females who have relatives living nearby are more likely to give alarm calls than females with no kin nearby. Males tend to call much less frequently, as would be expected because they are not related to most colony members. Another example of kin selection is provided by the whitefronted bee-eater, a bird that lives along river banks in Africa in colonies of 100 to 200 individuals (figure 53.32). In contrast to ground squirrels, male bee-eaters usually remain in the colony in which they were born, and the females disperse to join new colonies. Many bee-eaters do not raise their own offspring, but instead help others. Most helpers are young birds, but older birds whose nesting attempts have failed may also be helpers. The presence of a single helper, on average, doubles the number of offspring that survive. Two lines of evidence support the idea that kin selection is important in determining helping behavior in this species. First, helpers are normally males, which are usually related to other birds in the colony, and not females, which are not related. Second, when birds have the choice of helping different parents, they almost invariably choose the parents to which they are most closely related.

Figure 53.32 Kin selection in the white-fronted bee-eater, Merops bullockoides. Bee-eaters are small insectivorous birds that live in Africa in large colonies. Bee-eaters often help others raise their young; helpers usually choose to help close relatives. Heinrich van den Berg/ Getty Images

chapter

53 Behavioral Biology 1209

Learning Outcomes Review 53.12

Genetic and ecological factors have contributed to the evolution of altruism, a behavior that benefits another individual at a cost to the actor. Individuals may benefit directly if cooperative acts are reciprocated among unrelated interactants. Kin selection explains how altruistic acts directed toward relatives, which share alleles, increase an individual’s inclusive fitness. Haplodiploidy has resulted in eusociality among some insects by increasing genetic relatedness; it is not found in vertebrates. ■■

Imagine that you witness older group members rescuing infants in a troop of monkeys when a predator appears. How would you test whether the altruistic act you see is reciprocity or kin selection?

53.13  The Evolution of Group

Living and Animal Societies

Learning Outcomes 1. Explain the possible advantages of group living. 2. Contrast the nature of insect and vertebrate societies. 3. Discuss social organization in African weaver birds and how it is influenced by ecology.

Organisms from cnidarians and insects to fish, whales, chimpanzees, and humans live in social groups. To encompass the wide variety of social phenomena, we can broadly define a society as a group of organisms of the same species that are organized in a cooperative manner. Why have individuals in some species given up a solitary existence to become members of a group? One hypothesis is that individuals in groups benefit directly from social living. For example, a bird in a flock may be better protected from predators. As flock size increases, the risk of predation decreases because there are more individuals to scan the environment for predators. A member of a flock may also increase its feeding success if it can acquire information from other flock members about the location of new, rich food sources. In some predators, hunting in groups can increase success and allow the group to tackle prey too large for any one individual.

Insect societies form efficient colonies containing specialized castes In section 53.12, we discussed the origin of eusociality in the insect order Hymenoptera (ants, bees, and wasps). Additionally, all termites (order Isoptera) are also eusocial, and a few other insect and arthropod species are eusocial. Social insect colonies are composed of different castes, groups of individuals that differ in

1210 part VIII Ecology and Behavior

Figure 53.33 Castes of ants. These leaf-cutter ants are members of different castes. The large ant is a worker carrying leaves to the nest, whereas the smaller ant is protecting the worker from attack by small parasitic flies. Amazon-Images/Alamy Stock Photo

reproductive ability (queens versus workers), size, and morphology and perform different tasks. Workers nurse, maintain the nest, and forage; soldiers are large and have powerful jaws specialized for defense. The structure of an insect society is illustrated by leaf-cutters, which form colonies of as many as several million individuals. These ants cut leaves and use them to grow crops of fungi beneath the ground. Workers divide the tasks of leaf cutting, defense, mulching the fungus garden, and implanting fungal hyphae according to their body size (figure 53.33).

The structure of a vertebrate society is related to ecology In contrast to the highly structured and integrated insect societies and their remarkable forms of altruism, vertebrate social groups are usually less rigidly organized and less cohesive. It seems paradoxical that vertebrates, which have larger brains and are capable of more complex behaviors, are generally less altruistic than insects (the exception, of course, is humans). Reciprocity and kinselected altruism are common in vertebrate societies, although there is often more conflict and aggression among group members. Conflicts generally center on access to food and mates and occur because a vertebrate society is made up of individuals striving to improve their own fitness. Social groups of vertebrates have a group size, stability of members, number of breeding males and females, and type of mating system characteristic of a given species. Diet and predation are important factors in shaping social groups. For example, meerkats take turns watching for predators, while other group members forage for food (figure 53.34).

Figure 53.35 A naked mole rat. This and another mole rat species are the only known eusocial vertebrates. Mint Images/Science Source

Figure 53.34 Foraging and predator avoidance. A

meerkat sentinel on duty. Meerkats, Suricata suricata, are a species of highly social mongoose living in the semiarid sands of the Kalahari Desert in southern Africa. This meerkat is taking its turn to act as a lookout for predators. Under the security of its vigilance, the other group members can focus their attention on foraging.  Nigel Dennis/

Science Source

African weaver birds, which construct nests from vegetation, provide an excellent example of the relationship between ecology and social organization. Their roughly 90 species can be divided according to the type of social group they form. One group of species lives in the forest and builds camouflaged, solitary nests. Males and females are monogamous; they forage for insects to feed their young. The second group of species nests in colonies in trees on the savanna. They are polygynous and feed in flocks on seeds. The feeding and nesting habits of these two groups of species are correlated with their mating systems. In the forest, insects are hard to find, and both parents must cooperate in feeding the young. The camouflaged nests do not call the attention of predators to their brood. On the open savanna, building a hidden nest is not an option. Rather, savanna-dwelling weaver birds protect their young from predators by nesting in trees, which are not very abundant. This shortage of safe nest sites means that birds must nest together in colonies. Because seeds occur abundantly, a female can acquire all the food needed to rear young without a male’s help. The male,

free from the duties of parenting, spends his time courting many females—a polygynous mating system. One exception to the general rule that vertebrate societies are not organized like those of insects is the naked mole rat (Heterocephalus glaber), a small, hairless rodent that lives in and near East Africa (figure 53.35). Unlike other kinds of mole rats, which live alone or in small family groups, naked mole rats form large underground colonies with a far-ranging system of tunnels and a central nesting area. It is not unusual for a colony to contain 80 individuals. Naked mole rats feed on bulbs, roots, and tubers, which they locate by constant tunneling. As in insect societies, there is a division of labor among the colony members, with some individuals working as tunnelers, while others perform different tasks, depending on the size of their bodies. Large mole rats defend the colony and dig tunnels. Naked mole rat colonies have a reproductive division of labor similar to the one normally associated with the eusocial insects. All of the breeding is done by a single female, or “queen,” who has one or two male consorts. The workers, consisting of both sexes, keep the tunnels clear and forage for food.

Learning Outcomes Review 53.13

Advantages of group living include protection from predators and increased feeding success. Eusocial insects form complex, highly altruistic societies that increase the fitness of the colony. The members of vertebrate societies exhibit more conflict and competition, but also cooperate and behave altruistically, especially toward kin. African weaver birds have developed different types of societies depending on the ecology of their habitat, particularly the safety of nesting sites. ■■ ■■ ■■

What are the benefits and costs associated with living in social groups? Why is altruism directed toward kin considered to be selfish behavior? Is a human army more like an insect society or a vertebrate society? Explain your answer.

chapter

53 Behavioral Biology 1211

Chapter Review K. Ammann/Bruce Coleman Inc./Photoshot

53.1 The Natural History of Behavior Behavior can be analyzed in terms of mechanisms and evolutionary origin. Proximate causation refers to the mechanisms of behavior. Ultimate causation examines a behavior’s evolutionary significance. Ethology emphasizes the study of instinct and its origins. Innate, or instinctive, behavior is a response to an environmental stimulus or trigger that does not require learning (figure 53.1).

53.2 Nerve Cells, Neurotransmitters, Hormones,

and Behavior

Instinctive behaviors are accomplished by neural circuits, which develop under genetic control. Hormones and neurotransmitters can act to regulate behavior.

53.3 Behavioral Genetics Artificial selection, hybrid, and twin studies link genes and behavior. Breeding fast-learning and slow-learning rats for several generations produced two distinct behavioral populations (figure 53.3). Some behaviors appear to be controlled by a single gene.

Navigation is following a route based on orientation and some sort of “map.” The nature of the map in animals is not known.

53.8 Animal Communication Successful reproduction depends on appropriate signals and responses. Courtship signals are usually species-specific and help to ensure reproductive isolation (figure 53.17). Communication enables information exchange among group members (figures 53.18 and 53.19).

53.9 Behavior and Evolution Natural selection can lead to evolutionary change if variation exists, is genetically heritable, and leads to differences in fitness. Phylogenies can be used to understand the evolutionary history of behavior.

53.10 Behavioral Ecology Foraging behavior can directly influence energy intake and individual fitness. Natural selection favors optimal foraging strategies in which energy acquisition (cost) is minimized and reproductive success (benefit) is maximized.

53.4 Learning

Territorial behavior evolves if the benefits of holding a territory exceed the costs.

Learning mechanisms include habituation and association. Habituation, a form of nonassociative learning, is a decrease in response to repeated nonessential stimuli. Associative learning is a change in behavior by association of two stimuli or of a behavior and a response (conditioning). Classical (Pavlovian) conditioning occurs when two stimuli are associated with each other. Operant conditioning occurs when an animal associates a behavior with reward or punishment.

53.11 Reproductive Strategies

Instinct governs learning preparedness. What an animal can learn is biologically influenced—that is, learning is possible only within the boundaries set by evolution.

Mating systems reflect the ability of parents to care for offspring and are influenced by ecology. Mating systems include monogamy, polygyny, and polyandry; they are influenced by ecology and constrained by needs of offspring. Extra-pair copulations increase fitness in some monogamous species. For males, mating with more females leads to greater reproductive success. Females may benefit in several ways from mating with multiple males. Natural selection sometimes favors alternative mating strategies. Any behavior that confers greater fitness is likely to evolve.

53.5 The Development of Behavior

53.12 Altruism

Parent–offspring interactions influence how behavior develops. In imprinting, a young animal forms an attachment to other individuals or develops preferences that influence later behavior.

Reciprocity theory explains altruism between unrelated individuals. Mutual exchanges benefit both participants; a participant that does not reciprocate would not receive future aid.

Instinct and learning may interact as behavior develops. Animals may have an innate genetic template that guides their learning as behavior develops, such as song development in birds. Studies on twins reveal a role for both genes and environment in human behavior.

53.6 Animal Cognition

Kin selection theory proposes a direct genetic advantage to altruism. Kin selection increases the reproductive success of relatives and increased frequency of alleles shared by kin, and thus increases an individual’s inclusive fitness. Ants, bees, and wasps have haplodiploid reproduction, and therefore a high degree of gene sharing.

Some animals exhibit cognitive behavior and can devise solutions to novel situations (figures 53.10 and 53.11).

53.13  The Evolution of Group Living and Animal Societies

53.7 Orientation and Migratory Behavior

Insect societies form efficient colonies containing specialized castes (figure 53.33). Social insect societies are composed of different castes that are specialized to reproduce or to perform certain colony maintenance tasks.

Migration often involves populations moving large distances. Migrating animals must be capable of orientation and navigation (figure 53.14). Orientation is the mechanism by which animals move by tracking environmental stimuli such as celestial clues or Earth’s magnetic field. 1212 part VIII Ecology and Behavior

A social system is a group organized in a cooperative manner.

The structure of a vertebrate society is related to ecology. Vertebrate social systems are less rigidly organized and cohesive and are influenced by food availability and predation.

Visual Summary Behavior studied at level of

Mechanism includes

uses

include

in an individual includes

Nervous system

Association for Quick response Parental care

Hormones

Conditioning

uses

by when behavior has

Adaptive significance studied in

Orientation Navigation

includes

Behavioral ecology includes Foraging

Communication

Imprinting

Reproduction

Reproductive strategies

includes

Aggression Stress

Migration

Development of behavior

in

Genetics

Not just in primates

Learning

occurs

Natural selection

Cognition Habituation

Neural circuits

Evolution

Activities

Ontogeny

Instinct interact

studied t di d in i Artificial selection

Signals & responses

Mating systems

and Learning

Hybrids Twin studies

Courtship

Altruism

Identification & information exchange

Reciprocity Kin selection Animal societies

Waggle dance & alarm calls

Insects Vertebrates

Review Questions K. Ammann/Bruce Coleman Inc./Photoshot

U N D E R S TA N D 1. A key stimulus, innate releasing mechanism, and fixed action pattern a. b. c. d.

are mechanisms associated with behaviors that are learned. are components of behaviors that are innate. involve behaviors that cannot be explained in terms of ultimate causation. involve behaviors that are not subject to natural selection.

2. In operant conditioning a.

an animal learns that a particular behavior leads to a reward or punishment. b. an animal associates an unconditioned stimulus with a conditioned response. c. learning is unnecessary. d. habituation is required for an appropriate response.

chapter

53 Behavioral Biology 1213

3. The study of song development in sparrows showed that a. b. c. d.

the acquisition of a species-specific song is innate. there are two components to this behavior: a genetic template and learning. song acquisition is an example of associative learning. All of the choices are correct.

4. The difference between following a set of driving directions given to you by somebody on the street (for example “. . . take a right at the next light, go four blocks and turn left . . .”) and using a map to find your destination is a. b. c. d.

the difference between navigation and orientation, respectively. the difference between learning and migration, respectively. the difference between orientation and navigation, respectively. why birds are not capable of orientation.

5. In courtship communication a. b. c. d.

the signal itself is always species-specific. the sign communicates species identity. a stimulus–response chain is involved. courtship signals are produced only by males.

6. Behavioral ecology assumes a. b. c. d.

that all behavioral traits are innate. learning is the dominant determinant of behavior. behavioral traits are subject to natural selection. behavioral traits do not affect fitness.

11. Altruism a. b. c. d.

is possible only through reciprocity. is possible only through kin selection. can be explained only by group selection. will occur only when the fitness benefit of a given act is greater than the fitness cost.

A P P LY 1. Refer to figure 53.24. Data on size of mussels eaten by shore crabs suggest they eat sizes smaller than expected by an optimal foraging model. Suggest a hypothesis for why this is so and describe an experiment to test your hypothesis. 2. Refer to figure 53.25. Six pairs of birds were removed but only four pairs moved in. Where did the new pairs come from? Additionally, it appears that many of the birds that were not removed expanded their territories and that the new residents ended up with smaller territories than the pairs they replaced. Explain. 3. An altruistic act is defined as one that benefits another individual at a cost to the actor. There are two theories to explain how such behavior evolves: reciprocity and kin selection. How would you distinguish between the two in a field study? In the context of natural selection, is an altruistic act “costly” to an individual who performs it?

7. According to optimal foraging theory a. b. c. d.

individuals minimize energy intake per unit of time. energy content of a food item is the only determinant of a forager’s food choice. time taken to capture a food item is the only determinant of a forager’s food choice. a higher-energy item might be less valuable than a lowerenergy item if it takes too much time to capture the larger item.

8. From the perspective of females, extra-pair copulations (EPCs) a. b. c. d.

are always disadvantageous to females. can be associated with receiving male aid. are unimportant to fitness because they rarely lead to fertilization of eggs. can be of benefit only if the EPC male has elaborate secondary sexual traits.

9. In the haplodiploidy system of sex determination, males are a. b. c. d.

haploid. diploid. sterile. not present because bees exist as single-sex populations.

10. According to kin selection, saving the life of your _____ would do the least for increasing your inclusive fitness. a. mother b. brother

1214 part VIII Ecology and Behavior

c. sister-in-law d. niece

SYNTHESIZE 1. Insects that sting or contain toxic chemicals often have blackand-yellow coloration and consequently are not eaten by predators. How could you determine if a predator has an innate avoidance of insects that are colored this way, or if the avoidance is learned? If avoidance is learned, how would you determine the learning mechanism involved? How would you measure the adaptive significance of the black-and-yellow coloration to the prey insect? 2. Behavioral genetics has made great advances from detailed studies of a single animal such as the fruit fly as a model system to develop general principles of how genes regulate behavior. What are advantages and disadvantages of this “model system” approach? How would you determine how broadly applicable the results of such studies are to other animals? 3. If a female bird chooses to live in the territory of a particular male, why might she mate with a male other than the territory owner?

54 CHAPTER

Ecology of Individuals and Populations Chapter Contents 54.1

The Environmental Challenges

54.2 Populations: Groups of a Single Species in One Place 54.3 Population Demography and Dynamics 54.4 Life History and the Cost of Reproduction 54.5 Environmental Limits to Population Growth 54.6 Factors That Regulate Populations 54.7 Human Population Growth

Ian Cartwright/Getty Images

Introduction

Visual Outline Ecology of individuals and populations

Individuals respond to Insulation (°C cal/m2/h)

Populations

Environmental conditions

analyzed by examining

Population size

can limit

1.5

affected by

1.0

0.5

1.0

2.0

3.0

4.0

5.0

6.0

Thickness of Fur (mm)

Popu Population growth

Windblown Fruits

Demography & dynamics

Adherent Fruits

Life history & cost of reproduction

summarized in

Growth models

Regulating factors

7

Fleshy Fruits

6

5 Billions of People

Geographic distribution

4

3

2

1

4000 B.C.

3000 B.C.

2000 B.C.

1000 B.C.

Year

0

1000

2000

Ecology, the study of how organisms relate to one another and to their environments, is a complex and fascinating area of ­biology that has important implications for each of us. In our exploration of ecological principles, we first consider how organisms respond to the abiotic environment in which they exist and how these responses affect the properties of populations, emphasizing population dynamics. In chapter 55, we discuss communities of coexisting species and the interactions that occur among them. In chapters 56 through 58, we discuss the functioning of entire ecosystems and of the biosphere, concluding with a consideration of the problems facing our planet and our fellow species.

54.1 The

Environmental Challenges

Learning Outcomes 1. List some challenges that organisms face in their environments. 2. Describe ways in which individuals respond to environmental changes. 3. Explain how species adapt to environmental conditions.

The nature of the physical environment in large measure determines which organisms live in a particular climate or region. Key elements of the environment include: Temperature. Most organisms are adapted to live within a relatively narrow range of temperatures and will not thrive if temperatures are colder or warmer. The growing season of plants, for example, is importantly influenced by temperature. Water. All organisms require water. On land, water is often scarce, so patterns of rainfall have a major influence on life. Sunlight. Almost all ecosystems rely on energy captured by photosynthesis; the availability of sunlight influences the amount of life an ecosystem can support, particularly below the surface in marine communities. Soil. The physical consistency, pH, and mineral composition of the soil often severely limit terrestrial plant growth, particularly the availability of nitrogen and phosphorus. An individual encountering environmental variation may maintain a “steady-state” internal environment, a condition known as homeostasis. Many animals and plants actively employ physiological, morphological, or behavioral mechanisms to maintain hom­­e­os­­­tasis. The beetle in figure 54.1 is using a behavioral mechanism to

cope with limited water availability. Other animals and plants are known as conformers because they conform to the environment in which they find themselves, their bodies adopting the temperature, salinity, and other physical aspects of their surroundings. Responses to environmental variation can be seen over both the short and the long term. In the short term, spanning periods of a few minutes to an individual’s lifetime, organisms have a variety of ways of coping with environmental change. Over longer periods, natural selection can operate to make a population better adapted to its environment.

Organisms are capable of responding to environmental changes that occur during their lifetime During the course of a day, a season, or a lifetime, an individual organism must cope with a range of living conditions. Organisms do so through the physiological, morphological, and behavioral abilities they possess. These abilities are a product of natural selection acting in a particular environmental setting over time, which explains why an individual organism that is moved to a different environment may not survive.

Physiological responses Many organisms are able to respond to environmental change by making physiological adjustments. For example, you sweat when it is hot, increasing evaporative heat loss and thus preventing overheating. Similarly, people who visit high altitudes may initially experience altitude sickness—the symptoms of which include heart palpitations, nausea, fatigue, headache, mental impairment, and in serious cases, pulmonary edema—because of the lower atmospheric pressure and consequent lower oxygen availability in the air. After several days, however, the same people usually feel fine, because a number of physiological changes have increased the delivery of oxygen to their body tissues (table 54.1). Some insects avoid freezing in the winter by adding glycerol “antifreeze” to their blood; others tolerate freezing by converting much of their glycogen reserves into alcohols that protect their cell membranes from freeze damage.

Morphological capabilities Animals that maintain a constant internal temperature (endotherms) in a cold environment have adaptations that tend to minimize energy expenditure. For example, many mammals grow thicker coats during the winter, their fur acting as insulation to

TA B L E 5 4 .1

Physiological Changes at High Elevation

Increased rate of breathing

Figure 54.1 Meeting the challenge of obtaining moisture. On the dry sand dunes of the Namib Desert in

southwestern Africa, the fog-basking beetle, Onymacris unguicularis, collects moisture from the fog by holding its abdomen up at the crest of a dune to gather condensed water; water condenses as droplets and trickles down to the beetle’s mouth. Thorsten Milse/agefotostock 1216 part VIII Ecology and Behavior

Increased erythrocyte production, raising the amount of hemoglobin in the blood Decreased binding capacity of hemoglobin, increasing the rate at which oxygen is unloaded in body tissues Increased density of mitochondria, capillaries, and muscle myoglobin

Insulation (°C cal/m2/h)

1.5

winter summer wolf polar bear

1.0

0.5

1.0

2.0

3.0

4.0

5.0

6.0

Thickness of Fur (mm)

Figure 54.2 Morphological adaptation. Fur thickness in North American mammals has a major effect on the degree of insulation the fur provides.

does not have the opportunity to regulate its body temperature through behavioral means. Thus, it becomes a conformer and adopts the temperature of its ­surroundings. Behavioral adaptations can be extreme. Spadefoot toads (genus Scaphiophus), which are widely distributed in North America, can burrow nearly a meter below the surface and remain there for as long as nine months of each year, their metabolic rates greatly reduced as they live on fat reserves. When moist, cool conditions return, the toads emerge and breed. The young toads mature rapidly and then burrow back underground to await their turn to breed in the next year.

Natural selection leads to evolutionary adaptation to environmental conditions The ability of an individual to alter its physiology, morphology, or behavior is itself an evolutionary adaptation, the result of natural selection. The results of natural selection can also be detected by comparing populations of the same species that live in different environments. For example, as we’ve just seen, humans have the ability to acclimate physiologically to living at higher elevations. In addition, however, human populations can evolve adaptations for living at high altitudes; genetic research has shown that populations living at high altitudes have evolved genetic changes that make them better suited to such environments. For example, most Tibetan highlanders have two mutations in a gene related to oxygen uptake—but few individuals in nearby lowland populations have these mutations. These results suggest that natural selection has driven these mutations to high frequency

Body Temperature (°C)

retain body heat. In general, the thicker the fur, the greater the insulation (figure 54.2). Thus, a wolf’s fur is about three times thicker in winter than in summer and insulates more than twice as well. The ability of organisms to alter their morphology in response to environmental conditions is quite widespread. Plants, for example, will grow more extensive roots when water is limited and larger leaves in shaded environments. Animals, too, can alter their physique. In the presence of predators (usually detected by smell), snails grow thicker shells, daphnia develop spines, and tadpoles produce thicker tails (the better to swim quickly). The ability to produce multiple phenotypes from a single genotype is termed a norm of reaction. The existence of norms of reaction has several important consequences. Such flexibility is obviously useful to a species that may 32 find itself in different environments, particularly sedentary species that cannot just get up and move. 30 However, it is important to remember that such differences in phenotype among individuals do not 28 reflect genetic differences—rather, individuals may be genetically identical, but still have grown up 26 in different environments. Of course, a further conopen habitat sequence of this is that such nongenetic differences shaded forest 24 are not transmitted to the next generation because they are not genetically inherited. 24 26 28 30 32 Air Temperature (°C) If such flexibility is useful, why aren’t all traits so plastic? The presumed answer is that such flexibility has a cost such that individuals that are genetically Figure 54.3 Behavioral adaptation. In open habitats, the Puerto Rican crested programmed to produce only a single phenotype have lizard, Anolis cristatellus, maintains a relatively constant temperature by seeking out and an advantage that offsets the advantages of a large basking in patches of sunlight; as a result, it can maintain a relatively high temperature norm of reaction. This is an area of current research. even when the air is cool. In contrast, in shaded forests, this behavior is not possible, and

Behavioral responses Many animals deal with variation in the environment by moving from one patch of habitat to another, avoiding areas that are unsuitable. The tro­­­­­­­­­­­pical lizard in figure 54.3 manages to maintain a fairly uniform body temperature in an open habitat by basking in patches of sunlight to warm up and then retreating to the shade when it becomes too hot. By contrast, in shaded forests, the same lizard

the lizard’s body temperature conforms to that of its surroundings. (inset) Jonathan Losos

?

Inquiry question When given the opportunity, lizards regulate their

body temperature to maintain a temperature optimal for physiological functioning. Would lizards in open habitats exhibit different escape behaviors from those of lizards in shaded forest?

Data analysis Can the slope of the line tell us something about the behavior of the lizard?

chapter

54 Ecology of Individuals and Populations 1217

in very little time—the few thousand years since the Tibetans colonized the Himalayas. Researchers have recently discovered a number of other mutations in different genes involved in oxygen uptake and transport that also have evolved recently in these populations. This evidence is among the best examples of the action of natural selection on contemporary human populations. In addition to populations within a single species, closely related species often have evolved striking adaptations to the particular environment in which they live. For example, animals that live in different climates show many differences. Mammals from colder climates tend to have shorter ears and limbs—a phenomenon termed Allen’s rule—which reduces the surface area across which animals lose heat. Lizards that live in different climates exhibit physiological adaptations for coping with life at different temperatures. Desert lizards are unaffected by high temperatures that would kill a lizard from northern Europe, but the northern lizards are capable of running, capturing prey, and digesting food at cooler temperatures at which desert lizards would be completely i­ mmobilized. Many species also exhibit adaptations to living in areas where water is scarce. Everyone knows of the camel and other desert animals that can go extended periods without drinking water. Another example of desert adaptation is seen in frogs. Most frogs have moist skins through which water permeates readily. Such animals could not survive in arid climates because they would rapidly dehydrate and die. However, some frogs have solved this problem by evolving a greatly reduced rate of water loss through the skin. One species even secretes a waxy substance from specialized glands that waterproofs its skin and reduces rates of water loss by 95%. Adaptation to different environments can also be studied experimentally. For example, when strains of E. coli were grown at high temperatures (42°C), the speed at which the bacteria utilized resources improved through time. After 2000 generations, this ability increased 30% over what it had been when the experiment started.

Learning Outcomes Review 54.1

Environmental conditions include temperature, water and light availability, and soil characteristics. When the environment changes, individual organisms use a variety of physiological, morphological, and behavioral mechanisms to adjust. Over time, adaptations to different environments may evolve in populations. ■■

How might a species respond if its environment grew steadily warmer over time?

Organisms live as members of a population, groups of individuals that occur together at one place and time. In the rest of this chapter, we consider the properties of populations, focusing on factors that influence whether a population grows or shrinks, and at what rate. The explosive growth of the world’s human population in the last few centuries provides a focus for our inquiry. The term population can be defined narrowly or broadly. This flexibility allows us to speak in similar terms of the world’s human population, the population of protists in the gut of a termite, or the population of deer that inhabit a forest. Sometimes the boundaries defining a population are sharp, such as the edge of an isolated mountain lake for trout, and sometimes they are fuzzier, as when deer readily move back and forth between two forests separated by a cornfield. Three characteristics of population ecology are particularly important: (1) population range, the area throughout which a population occurs; (2) the pattern of spacing of individuals within that range; and (3) how the population changes in size through time.

A population’s geographic distribution is termed its range No population, not even one composed of humans, occurs in all habitats throughout the world. Most species, in fact, have relatively limited geographic ranges, and the range of some species is minuscule. For example, the Devil’s Hole pupfish lives in a single spring in southern Nevada (figure 54.4), and the Socorro isopod (Thermosphaeroma thermophilus) is known from a single spring system in New Mexico. At the other extreme, some species are widely distributed. The common dolphin (Delphinus delphis), for example, is found throughout all the world’s oceans. As discussed in section 54.1, organisms must be adapted to the environment in which they occur. Polar bears are exquisitely adapted to survive the cold of the Arctic, but you won’t find them in the tropical rainforest. Certain prokaryotes can live in the nearboiling waters of Yellowstone’s geysers, but they do not occur in cooler streams nearby. Each population has its own requirements— temperature, humidity, certain types of food, and a host of other factors—that determine where it can live and reproduce and where it can’t. In addition, in places that are otherwise suitable, the presence of predators, competitors, or parasites may prevent a population from occupying an area, a topic we will take up in chapter 55.

54.2 Populations:

Groups of a Single Species in One Place

Learning Outcomes 1. Distinguish between a population and a metapopulation. 2. Understand what causes a species’ geographic range to change through time.

1218 part VIII Ecology and Behavior

Figure 54.4 The Devil’s Hole pupfish. The Devil’s Hole pupfish, Cyprinodon diabolis, has the smallest range of any vertebrate species in the world. Stone Nature Photography/Alamy Stock Photo

Present 1966

Alpine tundra

Elevation (km)

3 km

Spruce-fir forests Mixed conifer forest

2 km

Woodlands

1 km

Grassland, chaparral, and desert scrub

1958

15,000 Years Ago

1951 1943

1937

1956

Alpine tundra Spruce-fir forests

Elevation (km)

Immigration from Africa

1961

Equator

2 km

1960

1965

0 km

3 km

1970

1964

1970

Mixed conifer forest

1 km

Woodlands

0 km

Grassland, chaparral, and desert scrub

Figure 54.5 Shifts in altitudinal distributions of trees in the mountains of southwestern North America. 

During the glacial period 15,000 years ago, conditions were cooler than they are now. As the climate warmed, tree species that require colder temperatures shifted their range upward in altitude so that they continue to live in the climatic conditions to which they are adapted.

Ranges undergo expansion and contraction Population ranges are not static but change through time. These changes occur for two reasons. In some cases, the environment changes. As the glaciers retreated at the end of the last ice age, approximately 10,000 years ago, many North American plant and animal populations expanded northward. At the same time, as climates warmed, species experienced shifts in the elevation at which they could live (figure 54.5). In addition, populations can expand their ranges when they are able to circumvent inhospitable habitat to colonize suitable, previously unoccupied areas. For example, the cattle egret is native to Africa. Some time in the late 1800s, these birds appeared in northern South America, having made the nearly 3500-km transatlantic crossing, perhaps aided by strong winds. Since then, they have steadily expanded their range and now can be found throughout most of the United States (figure 54.6).

The human effect By altering the environment, humans have allowed some species, such as coyotes, to expand their ranges and move into areas they

Figure 54.6 Range expansion of the cattle egret. The cattle egret (Bubulcus ibis)—so named because it follows cattle and other hoofed animals, catching any insects or small vertebrates they disturb—first arrived in South America from Africa in the late 1800s. Since the 1930s, the range expansion of this species has been well documented. The figure shows its spread over the first 100 or so years after arrival. Today, cattle egrets occur throughout almost all of South and Central America and the United States, as well as along the west coast of Canada. Shackleford-Photography/iStock/Getty Images previously did not occupy. Moreover, humans have served as an agent of dispersal for many species. Some of these transplants have been widely successful, as is discussed in greater detail in chapter 58. For example, 100 starlings were introduced into New York City in 1896 in a misguided attempt to establish every species of bird mentioned by Shakespeare. Their population steadily spread so that by 1980, they occurred throughout the United States. Similar stories could be told for countless plants and animals, and the list increases every year. Unfortunately, the success of these invaders often comes at the expense of native species.

Dispersal mechanisms Dispersal to new areas can occur in many ways. Lizards have colonized many distant islands, as one example, probably due to individuals or their eggs floating or drifting on vegetation. Bats are the only mammals on many distant islands because they can fly to them. chapter

54 Ecology of Individuals and Populations 1219

Windblown Fruits

Adherent Fruits

Fleshy Fruits

however, appear to exhibit random distributions in Panamanian rainforests.

Uniform spacing

Asclepias syriaca

Medicago polycarpa

Solanum dulcamara

Acer saccharum

Bidens frondosa

Juniperus chinensis

Terminalia calamansanai

Ranunculus muricatus

Rubus fruticosus

Figure 54.7 Some of the many adaptations for dispersal of seeds. Seeds have evolved a number of different

means of facilitating dispersal from their maternal plant. Some seeds can be transported great distances by the wind, whereas seeds enclosed in adherent or fleshy fruits can be transported by animals.

Seeds of plants are designed to disperse in many ways (figure 54.7). Some seeds are aerodynamically designed to be blown long distances by the wind. Others have structures that stick to the fur or feathers of animals, so that they are carried long distances before falling to the ground. Still others are enclosed in fleshy fruits. These seeds can pass through the digestive systems of mammals or birds and then germinate where they are defecated. Finally, seeds of mistletoes (Arceuthobium) are violently propelled from the base of the fruit in an explosive discharge. Although the probability of long-distance dispersal events leading to successful establishment of new populations is low, many such dispersals have occurred over millions of years.

Individuals in populations exhibit different spacing patterns Another key characteristic of population structure is the way in which individuals of a population are distributed. They may be randomly spaced, uniformly spaced, or clumped.

Random spacing Random spacing of individuals within populations occurs when they do not interact strongly with one another and when they are not affected by nonuniform aspects of their environment. Random distributions are not common in nature. Some species of trees,

1220 part VIII Ecology and Behavior

Uniform spacing within a population may often, but does not always, result from competition for resources. This spacing is accomplished, however, in many different ways. In animals, uniform spacing often results from behavioral interactions, as described in chapter 53. In many species, individuals of one or both sexes defend a territory from which other individuals are excluded. These territories provide the owner with exclusive access to resources, such as food, water, hiding refuges, or mates, and can space individuals evenly across the habitat if the resources themselves are evenly distributed. Even in nonterritorial species, individuals often maintain a defended space into which other animals are not allowed to intrude. Among plants, uniform spacing is also a common result of competition for resources. Closely spaced individual plants compete for available sunlight, nutrients, or water. These contests can be direct, as when one plant casts a shadow over another, or indirect, as when two plants compete by extracting nutrients or water from a shared area. In addition, some plants, such as the creosote bush, produce chemicals in the surrounding soil that are toxic to other members of their species. In all of these cases, only plants that are spaced an adequate distance from each other will be able to coexist, leading to uniform spacing.

Clumped spacing Individuals clump into groups or clusters in response to uneven distribution of resources in their immediate environments. Clumped distributions are common in nature because individual animals, plants, and microorganisms tend to occur in habitats defined by soil type, moisture, or other aspects of the environment to which they are best adapted. Social interactions also can lead to clumped distributions. Many species live and move around in large groups, which go by a variety of names (for example, flock, herd, pride). These groupings can provide many advantages, including increased awareness of and defense against predators, decreased energy cost of moving through air and water, and access to the knowledge of all group members. On a broader scale, populations are often most densely populated in the interior of their range and less densely distributed toward the edges. Such patterns usually result from the manner in which the environment changes in different areas. Populations are often best adapted to the conditions in the interior of their distribution. As environmental conditions change, individuals are less well adapted, and thus densities decrease. Ultimately, the point is reached at which individuals cannot persist at all; this marks the edge of a population’s range.

A metapopulation comprises distinct populations that may exchange members Species often exist as a network of distinct populations that exchange individuals. Such networks, termed ­ metapopulations, usually occur in areas in which suitable habitat is patchily

distributed and is separated by intervening stretches of unsuitable habitat.

Dispersal and habitat occupancy The degree to which populations within a metapopulation interact depends on the amount of dispersal. This interaction is often not symmetrical: populations increasing in size tend to send out many dispersers, whereas populations at low levels tend to receive more immigrants than they send off. In ­addition, relatively isolated populations tend to receive relatively few arrivals. Not all suitable habitats within a metapopulation’s area may be occupied at any one time. For a number of reasons, some individual populations may become extinct, perhaps as a result of an epidemic disease, a catastrophic fire, or the loss of genetic variation following a population bottleneck (see chapter 20). Dispersal from other populations, however, may eventually­recolonize such areas. In some cases, the number of habitats occupied in a metapopulation may represent an equilibrium in which the rate of extinction of existing populations is balanced by the rate of colonization of empty habitats.

Sweden occupied habitat patch unoccupied habitat patch

Norway

Finland Åland Islands

10 km

Source–sink metapopulations A species may also exhibit a metapopulation structure in areas in which some habitats are suitable for long-term population maintenance, but others are not. In these situations, termed source–sink metapopulations, the populations in the better areas (the sources) continually send out dispersers that bolster the populations in the poorer habitats (the sinks). In the absence of such continual replenishment, sink populations would have a negative growth rate and would eventually become extinct. Metapopulations of butterflies have been studied particularly intensively. In one study, researchers sampled populations of the Glanville fritillary butterfly at 1600 meadows in southwestern Finland (figure 54.8). On average, every year, 200 populations became extinct, but 114 empty meadows were colonized. A variety of factors seemed to increase the like­li­hood of a population’s extinction, including small population size, isolation from sources of immigrants, low resource availability (as indicated by the number of flowers on a meadow), and lack of genetic variation within the population. The researchers attribute the greater number of extinctions than colonizations to a string of very dry summers. Because none of the populations is large enough to survive on its own, continued survival of the species in southwestern Finland would appear to require the continued existence of a meta­pop­u­la­tion network in which new populations are continually created and existing populations are supplemented by immigrants. Continued bad weather thus may doom the species, at least in this part of its range. Metapopulations, where they occur, can have two important implications for the range of a species. First, through continuous colonization of empty patches, metapopulations prevent long-term extinction. If no such dispersal existed, then each population might eventually perish, leading to disappearance of the species from the entire area. Moreover, in source–sink metapopulations, the species

Figure 54.8 Metapopulations of butterflies. 

The Glanville fritillary butterfly, Melitaea cinxia, occurs in metapopulations in southwestern Finland on the Å land Islands. None of the populations is large enough to survive for long on its own, but continual immigration of individuals from other populations allows some populations to survive. In addition, continual establishment of new populations tends to offset extinction of established populations, although in recent years, extinctions have outnumbered colonizations.

occupies a larger area than it otherwise might, including marginal areas that could not support a population without a continual influx of immigrants. For these reasons, the study of metapopulations has become very important in conservation biology as natural habitats become increasingly fragmented.

Learning Outcomes Review 54.2

A population is a group of individuals of a single species existing together in an area. A population’s range, the area it occupies, changes over time. Populations, in turn, may form a network, or metapopulation, connected by individuals that move from one group to another. Within a population, the distribution of individuals can be random, uniform, or clumped, and the distribution is determined in part by the availability of resources. ■■

How might the geographic range of a species change if populations could not exchange individuals with each other? How would your answer depend on what type of metapopulation existed?

chapter

54 Ecology of Individuals and Populations 1221

males in species in which a single male can mate with several females. In many species, males compete for the opportunity to mate with females, as you learned in chapter 20; consequently, a few males have many matings, and many males do not mate at all. In such species, the sex ratio of reproducing animals is female-biased and does not affect population growth rates; reduction in the number of males simply changes the identities of the reproductive males without reducing the number of births. By contrast, among monogamous species, pairs may form long-lasting reproductive relationships, and a reduction in the number of males can then directly reduce the number of births. Generation time is the average interval between the birth of an individual and the birth of its offspring. This factor can also affect population growth rates. Species differ greatly in generation time. Differences in body size can explain much of this variation— mice go through approximately 100 generations during the course of one elephant generation (­figure 54.9). But small size does not always mean short generation time. Newts, for example, are smaller than mice, but have considerably longer generation times. In general, populations with short generations can increase in size more quickly than populations with long generations. Conversely, because generation time and life span are usually closely correlated, populations with short generation times may also diminish in size more rapidly if birth rates suddenly decrease.

54.3 Population

Demography and Dynamics

Learning Outcomes 1. Define demography. 2. Describe the factors that influence a species’ demography. 3. Explain the significance of survivorship curves.

The dynamics of a population—how it changes through time—are affected by many factors. One important factor is the age distribution of individuals—that is, what proportion of individuals are adults, juveniles, and young. Demography is the quantitative study of populations. How the size of a population changes through time can be studied at two levels: as a whole or broken down into parts. At the most inclusive level, we can study the whole population to determine whether it is increasing, decreasing, or remaining constant. Put simply, populations grow if births outnumber deaths and shrink if deaths outnumber births (ignoring, for the moment, immigration and extinction). Understanding these trends is often easier, however, if we break the population into smaller units composed of individuals of the same age (for example, 1-year-olds) and study the factors affecting birth and death rates for each unit separately.

Age structure is determined by the numbers of individuals in different age groups A group of individuals of the same age is referred to as a cohort. In most species, the probability that an individual will reproduce or die varies through its life span. As a result, within a population, every cohort has a characteristic birth rate, or fecundity rate, defined as the number of offspring produced in a standard time (for example, per year), and a characteristic death rate, or m ­ ortality rate, the number of individuals that die in that period.

Sex ratio and generation time affect population growth rates Population growth can be influenced by the population’s sex ratio. The number of births in a population is usually directly related to the number of females; births may not be as closely related to the number of

1 week

1 hour

1222 part VIII Ecology and Behavior

Daphnia Stentor Spirochaeta Euglena

Paramecium Didinium Tetrahymena

Pseudomonas

Body Size (length)

100 m

10 m

1m

10 cm

E. coli

1 μm

How well could you predict generation time from body size?

1 month

1 cm

Data analysis 

1 year

1 day

Sequoia Birch Fir Elephant Balsam

Horseshoe crab Turtle Dogwood Whale Newt Snake Bear Rhino Chameleon Beaver Clam Kelp Frog Deer Elk Crab Oyster Fox Snail Scallop Rat Horsefly Salamander Mouse Housefly Bee Drosophila

10 years

1 mm

If resources became more abundant, would you expect smaller or larger species to increase in population size more quickly?

Human

100 μm

?

Inquiry question 

100 years

10 μm

In general, larger organisms have longer generation times, although there are exceptions.

Generation Time

Figure 54.9 The relationship between body size and generation time. 

The relative number of individuals in each cohort defines a population’s age structure. Because different cohorts have different fecundity and death rates, age structure has a critical influence on a population’s growth rate. Populations with a large proportion of young individuals, for example, tend to grow rapidly because an increasing proportion of their individuals are reproductive. Human populations in many developing countries are an example, as will be discussed in section 54.7. Conversely, if a large proportion of a population is relatively old, populations may decline. This phenomenon now characterizes Japan and some countries in Europe.

Life tables show probability of survival and reproduction through a cohort’s life span To assess how populations in nature are changing, ecologists use a life table, which tabulates the fate of a cohort from birth until death, showing the number of offspring produced and the number of individuals that die in each time period. Table 54.2 is an example of a life table analysis from a study of the annual bluegrass. This study follows the fate of 843 individuals through time, charting how many survive in each interval and how many offspring each survivor produces. In table 54.2, the first column indicates the age of the cohort (that is, the number of 3-month intervals from the start of the study). The second and third columns indicate the number of survivors and the proportion of the original cohort still alive at the beginning of that interval. The fifth column presents the mortality rate, the proportion of individuals that started that interval alive but died by the end of it. The seventh column indicates the average number of seeds produced by each surviving individual in that interval, and the last column shows the number of seeds produced relative to the size of the original cohort. Much can be learned by examining life tables. In the case of the annual bluegrass, we see that both the probability of dying and

TA B L E 5 4 . 2

Age (in 3-month intervals)

the number of offspring produced per surviving individual steadily increases with age. By adding up the numbers in the last column, we get the total number of offspring produced per individual in the initial cohort. This number is almost 2, which means that for every original member of the cohort, on average two new individuals have been produced. A value of 1.0 would be the break-even number, the point at which the population was neither growing nor shrinking. In this case, the population appears to be growing rapidly.

Data analysis If annual bluegrass were an undesirable weed and you wanted to target one age group to remove, would you focus on the age class that produces the most seeds per individual? Can you imagine any circumstance in which your answer would be different? In most cases, life table analysis is more complicated than this. First, except for organisms with short life spans, it is difficult to track the fate of a cohort until the death of the last individual. An alternative approach is to construct a cross-­sectional study, examining the fate of cohorts of different ages in a single period. In addition, many factors—such as offspring reproducing before all members of their parents’ cohort have died—complicate the interpretation of whether populations are growing or shrinking.

Survivorship curves demonstrate how survival probability changes with age The percentage of an original population that survives to a given age is called its survivorship. One way to express some aspects of the age distribution of populations is through a survivorship curve. Examples of different survivorship curves are shown in figure 54.10. Oysters produce vast numbers of offspring, only a

Life Table of the Annual Bluegrass (Poa annua) for a Cohort Containing 843 Seedlings Number Alive at Beginning of Time Interval

Proportion of Cohort Alive at Beginning of Time Interval (survivorship)

Deaths During Time Interval

Mortality Rate During Time Interval

Seeds Produced During Time Interval

Seeds Produced per Surviving Individual (fecundity)

Seeds Produced per Member of Cohort (fecundity × survivorship)

0

843

1.000

121

0.144

0

0.00

0.00

1

722

0.856

195

0.270

303

0.42

0.36

2

527

0.625

211

0.400

622

1.18

0.74

3

316

0.375

172

0.544

430

1.36

0.51

4

144

0.171

90

0.626

210

1.46

0.25

5

54

0.064

39

0.722

60

1.11

0.07

6

15

0.018

12

0.800

30

2.00

0.04

7

3

0.004

3

1.000

10

3.33

0.01

8

0

0.000

—

Total = 1665

chapter

Total = 1.98

54 Ecology of Individuals and Populations 1223

54.4 Life

History and the Cost of Reproduction

1.0

Proportion Surviving

Human (type I)

Hydra (type II)

0.1

Learning Outcomes 1. Describe reproductive trade-offs in an organism’s life history. 2. Compare the costs and benefits of allocating resources to reproduction.

0.01 Oyster (type III) 0.001

0

25 50 75 Percent of Maximum Life Span

Natural selection favors traits that maximize the number of surviving offspring left in the next generation by an individual organism. Two factors affect this quantity: how long an individual lives, and how many young it produces each year. Why doesn’t every organism reproduce immediately after its own birth, produce large families of offspring, care for them intensively, and perform these functions repeatedly throughout a long life? The answer is that usually no one organism can do all of this, simply because not enough resources are available. Consequently, organisms allocate resources either to current reproduction or to increasing their prospects of surviving and reproducing at later life stages. The complete life cycle of an organism constitutes its life history. All life histories involve significant trade-offs. Because

100

Figure 54.10 Survivorship curves. By convention, survival (the vertical axis) is plotted on a log scale, which produces a straight line if the rate of mortality is constant through time. Humans have a type I life cycle, hydra (an animal related to jellyfish) type II, and oysters type III.

1.0

Proportion Surviving

few of which live to reproduce. However, once they become established and grow into reproductive individuals, their mortality rate is extremely low (type III survivorship curve). In hydra, animals related to jellyfish, individuals are equally likely to die at any age. The result is a straight survivorship curve (type II). Finally, mortality rates in humans, as in many other animals and in protists, rise steeply later in life (type I survivorship curve). Of course, these descriptions are just generalizations, and many organisms show more complicated patterns. Examination of the data for annual bluegrass, for example, reveals that it is most similar to a type II survivorship curve (figure 54.11).

■■

Will populations with higher survivorship rates always have higher population growth rates than populations with lower survivorship rates? Explain.

1224 part VIII Ecology and Behavior

0.1 0.05 0.04 0.03 0.02 0.01 0.005 0.004 0.003 0.002

Learning Outcomes Review 54.3

Demography is the quantitative study of populations. Demographic characteristics include age structure, life span, sex ratio, generation time, and birth and mortality rates. The age structure of a population and the manner in which mortality and birth rates vary among different age cohorts determine whether a population will increase or decrease in size.

0.5 0.4 0.3 0.2

3

6

9

12

15

18

21

24

27

Age (months)

Figure 54.11  Survivorship curve for a cohort of annual bluegrass. After several months of age, mortality occurs at a constant rate through time.

?

Inquiry question Suppose you wanted to keep

annual bluegrass in your room as a houseplant. Suppose, too, that you wanted to buy an individual plant that was likely to live as long as possible. What age plant would you buy? How might the shape of the survivorship curve affect your answer?

Number of Offspring per Year

5

7

6

5

−2 −1

0

+1 +2

Change in Clutch Size

Figure 54.13 Reproductive events per lifetime. Adding

eggs to nests of collared flycatchers, Ficedula albicollis, which increases the reproductive efforts of the female rearing the young, decreases clutch size the following year; removing eggs from the nest increases the next year’s clutch size. This experiment demonstrates the trade-off between current reproductive effort and future reproductive success. (left) ©Christian Kerihuel

Data analysis Which has a greater effect, removing eggs from or adding eggs to a nest?

Alternatively, when costs of reproduction are high, lifetime reproductive success may be maximized by deferring or minimizing current reproduction to enhance growth and survival rates. This situation may occur when costs of reproduction significantly affect the ability of an individual to survive or decrease the number of offspring that can be produced in the future.

2

A trade-off exists between number of offspring and investment per offspring

1 0.5 0.2 0.1 0.05

0.1

0.2

0.5

1.0

Adult Mortality Rate per Year

Figure 54.12 Reproduction has a price. Data from many

bird species indicate that increased fecundity in birds correlates with higher mortality, ranging from the albatross (lowest) to the sparrow (highest). Species that raise more offspring per year have a higher probability of dying during that year.

?

Clutch Size Following Year

resources are limited, a change that increases reproduction may decrease survival and reduce future reproduction. As one example, a Douglas fir tree that produces more cones increases its current reproductive success—but it also grows more slowly. Because the number of cones produced is a function of how large a tree is, this diminished growth will decrease the number of cones it can produce in the future. Similarly, birds that have more offspring each year have a higher probability of dying during that year or of producing smaller clutches the following year (figure 54.12). Conversely,­individuals that delay reproduction may grow faster and larger,­enhancing future reproduction. In one elegant experiment, researchers changed the number of eggs in the nests of a bird, the collared flycatcher (figure 54.13). Birds whose clutch size (the number of eggs produced in one breeding event) was decreased expended less energy raising their young and thus were able to lay more eggs the next year, whereas those given more eggs worked harder and conse­quently produced fewer eggs the following year. Ecologists refer to the reduction in future reproductive potential resulting from current reproductive efforts as the cost of reproduction. Natural selection favors the life history that maximizes lifetime reproductive success. When the cost of reproduction is low, individuals should produce as many offspring as possible because there is little cost. Low costs of reproduction may occur when resources are abundant and may also be relatively low when overall mortality rates are high. In the latter case, individuals may be unlikely to survive to the next breeding ­season anyway, so the incremental effect of increased reproductive efforts may have little effect on future survival.

Inquiry question One explanation for this relationship

is that birds that expend energy to raise more offspring in one year are more likely to die. What is an alternative explanation based on evolutionary adaptation?

In terms of natural selection, the number of offspring produced is not as important as how many of those offspring themselves survive to reproduce. Assuming that the amount of energy to be invested in offspring is limited, a balance must be reached between the number of offspring produced and the size of each offspring (figure 54.14). This trade-off has been experimentally demonstrated in the side-blotched lizard, which normally lays between four and five eggs at a time. When some of the egg follicles are removed surgically early in the reproductive cycle, the female lizard produces only one to three eggs, but supplies each of these eggs with greater amounts of yolk, producing eggs and, subsequently, hatchlings that are much larger than normal (figure 54.15). In the side-blotched lizard and many other species, the size of offspring is critical—larger offspring have a greater chance of survival. Producing many offspring with little chance of survival might not be the best strategy, but producing only a single, extraordinarily robust offspring also would not maximize the chapter

54 Ecology of Individuals and Populations 1225

Nestling Size (g)

19.5 19.0

18.5 18.0 17.5 0

2

4

6

8

10

12

14

Clutch Size

Figure 54.14 The relationship between clutch size and offspring size. 

In great tits, Parus major, the size of the nestlings is inversely related to the number of eggs laid. The more mouths they have to feed, the less the parents can provide to any one nestling.

Data analysis Overall, what clutch size probably requires the greatest energy expenditure by the parents?

?

Inquiry question Would natural selection favor producing many small young or a few large ones?

number of surviving offspring. Rather, an intermediate situation, in which several fairly large offspring are produced, should lead to the maximum number of surviving offspring.

Reproductive events per lifetime represent an additional trade-off The trade-off between age and fecundity plays a key role in many life histories. Annual plants and most insects focus all their reproductive resources on a single large event and then die. This life history adaptation is called semelparity. Organisms that produce offspring several times over many seasons exhibit a life history adaptation called iteroparity. Species that reproduce yearly must avoid overtaxing themselves in any one reproductive episode so that they will be able to survive and reproduce in the future. Semelparity, or “big bang” reproduction, is usually found in short-lived species that have a low probability of staying alive to reproduce again, such as plants growing in harsh climates. Semelparity is also favored when fecundity entails large reproductive cost, exemplified by Pacific salmon migrating upriver to their spawning grounds. In these species, rather than investing some resources in an unlikely bid to survive until the next breeding season, individuals put all their resources into one reproductive event. 1226 part VIII Ecology and Behavior

Figure 54.15 Variation in the size of baby sideblotched lizards, Uta stansburiana, produced by experimental manipulations.  In clutches in which some

developing eggs were surgically removed, the remaining offspring were larger (center) than lizards produced in control clutches in which all the eggs were allowed to develop (right). The causal relationship between yolk provided by the mother and size of the offspring was demonstrated in experiments in which some of the yolk was removed from the eggs and smaller lizards resulted (left). Barry Sinervo

Age at first reproduction correlates with life span Among mammals and many other animals, longer-lived species put off reproduction longer than short-lived species, relative to expected life span. The advantage of delayed reproduction is that juveniles gain experience and can grow more rapidly before expending the high costs of reproduction. In long-lived animals, these advantages outweigh the costs in lost reproduction of not breeding earlier. In shorter-lived animals, on the other hand, time is of the essence; thus, quick reproduction is more critical than juvenile training and faster growth, and reproduction tends to occur earlier.

Learning Outcomes Review 54.4

Life history adaptations involve many trade-offs between reproductive cost and investment in survival. These trade-offs take a variety of forms, from laying fewer than the maximum possible number of eggs to putting all energy into a single bout of reproduction. Natural selection favors maximizing lifetime reproductive success. ■■

How might the life histories of two species differ if one were subject to high levels of predation and the other had few predators?

54.5 Environmental

Learning Outcomes 1. Explain exponential growth. 2. Discuss why populations cannot grow exponentially forever. 3. Define carrying capacity and explain what might cause it to change.

Populations often remain at a relatively constant size, regardless of how many offspring are born. As you saw in chapter 1, Darwin based his theory of natural selection partly on this seeming contradiction. Natural selection occurs because of checks on reproduction, with some individuals producing fewer surviving offspring than others. To understand populations, we must consider how they grow and what factors in nature limit population growth.

The exponential growth model applies to populations with no growth limits The rate of population increase, r, is defined as the difference between the birth rate, b, and the death rate, d, corrected for movement of individuals into or out of the population (e, rate of movement out of the area; i, rate of movement into the area). Thus, r = (b – d ) + (i – e) Movements of individuals can have a major influence on population growth rates. For example, the increase in human population in the United States during the closing decades of the 20th century was mostly due to immigration. The simplest model of population growth assumes that a population grows without limits at its maximal rate and also that rates of immigration and emigration are equal. This rate, called the biotic potential, is the rate at which a population of a given species increases when no limits are placed on its rate of growth. In mathematical terms, this is defined by the following formula: dN = r N i dt where N is the number of individuals in the population, dN/dt is the rate of change in its numbers over time, and ri is the intrinsic rate of natural increase for that population—its innate capacity for growth. The biotic potential of any population is exponential (red line in figure 54.16). Even when the rate of increase remains constant, the actual number of individuals accelerates rapidly as the size of the population grows. The result of unchecked exponential growth is a population explosion. A single pair of houseflies, laying 120 eggs per generation, could produce more than 5 trillion descendants in a year. In 10 years, their descendants would form a swarm more than 2 m thick over the entire surface of the Earth! In practice, such patterns

1250

Population Size (N)

Limits to Population Growth

dN = 1.0 N dt

Carrying capacity

1000 750 dN = 1.0 N dt

500

1000 – N 1000

250 0

0

5 10 Number of Generations (t)

15

Figure 54.16 Two models of population growth. 

The red line illustrates the exponential growth model for a population with an r of 1.0. The blue line illustrates the logistic growth model in a population with r = 1.0 and K = 1000 individuals. At first, logistic growth accelerates exponentially; then, as resources become limited, the death rate increases and growth slows. Growth ceases when the death rate equals the birth rate. The carrying capacity (K) ultimately depends on the resources available in the environment.

Data analysis Suppose in one species of mouse, each pair produced four surviving offspring, whereas in another population, each pair produced five offspring. If this rate of population increase remained unchanged, after 10 generations, how much larger would the population of the second species be?

of unrestrained growth prevail only for short periods, usually when an organism reaches a new habitat with abundant resources. Natural examples of such a short period of unrestrained growth include dandelions arriving in the fields, lawns, and meadows of North America from Europe for the first time; algae colonizing a newly formed pond; or cats introduced to an island with many birds that were previously lacking predators.

Carrying capacity No matter how rapidly populations grow, they eventually reach a limit imposed by shortages of important environmental factors, such as space, light, water, or nutrients. A population ultimately may stabilize at a certain size, called the carrying capacity of the particular place where it lives. The carrying capacity, symbolized by K, is the maximum number of individuals that the environment can support.

The logistic growth model applies to populations that approach their carrying capacity As a population approaches its carrying capacity, its rate of growth slows greatly, because fewer resources remain for each new individual to use. The growth curve of such a population, chapter

54 Ecology of Individuals and Populations 1227

In this model of population growth, the growth rate of the population (dN/dt) is equal to its intrinsic rate of natural increase (r multiplied by N, the number of individuals present at any one time), adjusted for the amount of resources available. The adjustment is made by multiplying rN by the fraction of K, the carrying capacity, still unused [(K – N )/K   ]. As N increases, the fraction of resources by which r is multiplied becomes smaller and smaller, and the rate of increase of the population declines. Graphically, if you plot N versus t (time), you obtain a ­sigmoidal growth curve characteristic of many biological populations. The curve is called “sigmoidal” because its shape has a double curve like the letter S. As the size of a population stabilizes at the carrying capacity, its rate of growth slows, eventually coming to a halt (blue line in figure 54.16). In mathematical terms, as N approaches K, the rate of population growth (dN/dt) begins to slow, reaching 0 when N = K (figure 54.17). Conversely, if the population size exceeds the carrying capacity, then K – N will be negative, and the population will experience a negative growth rate. As the population size then declines toward the carrying capacity, the magnitude of this negative growth rate will decrease until it reaches 0 when N = K. Notice that the population tends to move toward the carrying capacity regardless of whether it is initially above or below it. For this reason, logistic growth tends to return a population to the same size. In this sense, such populations are considered to be in equilibrium because they would be expected to be at or near the carrying capacity at most times. In many cases, real populations display trends corresponding to a logistic growth curve. This is true not only in the laboratory, but also in natural populations (figure 54.18a). In some cases, however, the fit is not perfect (figure 54.18b), and as we shall see shortly, many populations exhibit other patterns.

Callorhinus ursinus, population on St. Paul Island, Alaska. b. Two laboratory populations of the cladoceran, Bosmina longirostris. Note that the populations first exceeded the carrying capacity, before decreasing to what appears to be a size that was then maintained.

Number of Breeding Male Fur Seals (thousands)

Figure 54.18 Many populations exhibit logistic growth. a. A fur seal,

Positive Growth Rate

Negative Growth Rate Below K

1228 part VIII Ecology and Behavior

Above K

Figure 54.17 Relationship between population growth rate and population size. Populations far from the carrying

capacity (K) have high growth rates—positive if the population is below K, and negative if it is above K. As the population approaches K, growth rates approach zero.

?

Inquiry question Why does the growth rate converge on zero?

Learning Outcomes Review 54.5

Exponential growth refers to population growth in which the number of individuals accelerates even when the rate of increase remains constant; it results in a population explosion. Exponential growth is eventually limited by resource availability. The size at which a population in a particular location stabilizes is defined as the carrying capacity of that location for that species. Populations often grow to the carrying capacity of their environment. ■■

What might cause a population’s carrying capacity to change, and how would the population respond?

8 6 4 2 0

a.

Carrying Capacity (K) Population Size (N)

10

1915

N=K

0

1925 1935 Time (years)

1945

Number of Cladocerans (per 200 mL)

( )

dN = rN K – N dt K

Population Growth Rate per Capita

which is always limited by one or more factors in the environment, can be approximated by the following logistic growth equation:

b.

500 400 300 200 100 0 0

10

20 30 40 Time (days)

50

Learning Outcomes 1. Compare density-dependent and density-independent factors. 2. Evaluate why the size of some populations cycles. 3. Consider how the life history adaptations of species may differ depending on how often populations are at their carrying capacity.

A number of factors may affect population size through time. Some of these factors depend on population size and are therefore termed density-dependent. Other factors, such as natural disasters, affect populations regardless of size; these factors are termed density-independent. Many populations exhibit cyclic fluctuations in size that may result from complex interactions of factors.

5.0

0.9

4.0

0.8 0.7

3.0

0.6

2.0

0.5

1.0

0.4 0

20

40 80 120 60 100 Number of Breeding Adults

Juvenile Mortality

That Regulate Populations

Number of Young per Female

54.6 Factors

140

Figure 54.20 Density dependence in the song sparrow, Melospiza melodia, on Mandarte Island.  Reproductive success decreases and mortality rates increase as population size increases.

?

Inquiry question What would happen if researchers supplemented the food available to the birds?

High

High

High

Low

Low

Low

which also results in increasing rates of mortality as populations increase. High population densities can also lead to an accumulaDensity-dependent effects occur when tion of toxic wastes in the environment. reproduction and survival are affected Behavioral changes may also affect population growth rates. by population size Some species of rodents, for example, become antisocial, fighting more, breeding less, and generally acting stressed-out. These behavThe reason population growth rates are affected by population size ioral changes result from hormonal actions, but their ultimate cause is is that many important processes have density-­dependent effects. not yet clear; most likely, they have evolved as adaptive responses to That is, as population size increases, either reproductive rates desituations in which resources are scarce. In addition, in crowded cline or mortality rates increase, or both, a phenomenon termed populations, the population growth rate may decrease because of an negative feedback (figure 54.19). increased rate of emigration of individuals attempting to find better Populations can be regulated in many different ways. conditions elsewhere (figure 54.21). When populations approach their carrying capacity, competition However, not all density-dependent factors are negatively for resources can be severe, leading to both a decreased birth rate related to population size. In some cases, growth rates increase and an increased risk of death (­figure 54.20). In addition, predators with population size. This phenomenon is referred to as the Allee often focus their attention on a particularly common prey species, effect (after American zoologist Warder Allee, who first described it), and is an example of positive feedback. The Allee effect can take several Affecting Birth Rates Affecting Death Rates Affecting Birth and Death Rates forms. Most obviously, in populations that are too DensityDensityDensitydependent sparsely distributed, indidependent dependent death rate birth rate birth rate viduals may have difficulty Densityindependent finding mates. Moreover, DensityDensitydeath rate independent dependent some species may rely on birth rate death rate large groups to deter predators or to provide the Equilibrium Equilibrium Equilibrium population necessary stimulation for population population density density breeding activities. The density Allee effect is a major threat Population Density Population Density Population Density for many endangered species, which may never reFigure 54.19 Density-dependent population regulation. Density-dependent factors can affect cover from decreased birth rates, death rates, or both. population sizes caused by habitat destruction, overexploitation, or other causes Inquiry question Why might birth rates be density-dependent? (see chapter 58).

?

chapter

54 Ecology of Individuals and Populations 1229

103

Panolis

Figure 54.21 Density-dependent effects. Migratory

locusts, Locusta migratoria, are a legendary plague of large areas of Africa and Eurasia. At high population densities, the locusts have different hormonal and physical characteristics and take off as a swarm. (left) ruvanboshoff/E+/Getty images; (right) Juniors Tierbildarchiv/

Photoshot

Number of Moth Pupae per m2 Plotted on a Logarithmic Scale

10 Hyloicus

103 10 105

Dendrolimus

103 10 105

Bupalus

103 10 1880

1890

1900

1910

1920

1930

1940

Year

Density-independent effects include environmental disruptions and catastrophes Growth rates in populations sometimes do not correspond to the logistic growth equation. In many cases, such patterns result because growth is under the control of density-­independent effects. In other words, the rate of growth of a population at any instant is limited by something unrelated to the size of the population. A variety of factors may affect populations in a densityindependent manner. Most of these are aspects of the external environment, such as extremely cold winters, droughts, storms, or volcanic eruptions. Individuals often are affected by these occurrences regardless of the size of the population. Populations in areas where such events occur relatively frequently display erratic growth patterns in which the populations increase rapidly when conditions are benign, but exhibit large reductions whenever the environment turns hostile (figure 54.22). Needless to say, such populations do not produce the sigmoidal growth curves characteristic of the logistic equation.

Population cycles may reflect complex interactions In some populations, density-dependent effects lead not to an equilibrium population size but to cyclic patterns of increase and decrease. For example, ecologists have studied cycles in hare populations since the 1820s. They have found that the North American snowshoe hare (Lepus americanus) follows a “10-year cycle” (in reality, the cycle varies from 8 to 11 years). Hare population numbers fall 10-fold to 30-fold in a typical cycle, and 100-fold changes can occur (figure 54.23). Two factors appear to be generating the cycle—food plants and predators: Food plants. The preferred foods of snowshoe hares are willow and birch twigs. As hare density increases, the quantity of these twigs decreases, forcing the hares to feed on 1230 part VIII Ecology and Behavior

Figure 54.22 Fluctuations in the number of pupae of four moth species in Germany. The

population fluctuations suggest that density-independent factors are regulating population size. The concordance in trends through time suggests that the same factors are regulating population size in all four species.

high-fiber (low-quality) food. Lower birth rates, low juvenile survivorship, and low growth rates follow. The hares also spend more time searching for food, an activity that increases their exposure to predation. The result is a precipitous decline in willow and birch twig abundance, and a corresponding fall in hare abundance. It takes two to three years for the quantity of mature twigs to recover. Predators. A key predator of the snowshoe hare is the Canada lynx. The Canada lynx shows a “10-year” cycle of abundance that seems remarkably synchronized to the hare abundance cycle (figure 54.23). As hare numbers increase, lynx numbers do too, rising in response to the increased availability of the lynx’s food. When hare numbers fall, so do lynx numbers, their food supply depleted. Which factor is responsible for the predator–prey oscillations? Do increasing numbers of hares lead to overharvesting of plants (a hare–plant cycle), or do increasing numbers of lynx lead to over­ harvesting of hares (a hare–lynx cycle)? Field experiments carried out by Charles Krebs and coworkers provide an answer. In Canada’s Yukon, Krebs set up experimental plots that contained hare populations. If food is added (no food shortage effect) and predators are excluded (no predator effect) in an experimental area, hare numbers increase 10-fold and stay there—the cycle is lost. However, the cycle is retained if either of the factors is allowed to operate alone: exclude predators but don’t add food (food shortage effect alone), or add food in the presence of predators (predator effect alone). Thus, both factors can affect the cycle, which in practice seems to be generated by the interaction between the two.

Number of Pelts (in thousands)

160 120

snowshoe hare lynx

80 40 0 1845 1855 1865 1875 1885 1895 1905 1915 1925 1935 Year

Figure 54.23 Linked population cycles of the snowshoe hare, Lepus americanus, and the northern lynx, Lynx canadensis. These data are based on records of fur returns from trappers in the Hudson Bay region of Canada. The lynx population carefully tracks that of the snowshoe hare, but lags behind it slightly. Alan & Sandy Carey/Science Source

?

Inquiry question Suppose experimenters artificially kept the hare population at a high and constant level; what would happen to the lynx population? Conversely, if experimenters artificially kept the lynx population at a high and constant level, what would happen to the hare population?

sizes fluctuate markedly and are often far below carrying capacity. The selective factors affecting such species differ markedly. Individuals in populations near their carrying capacity may face stiff competition for limited resources; by contrast, individuals in populations far below carrying capacity have access to abundant resources. When resources are limited, the cost of reproduction often will be very high. Consequently, selection will favor individuals that can compete effectively and utilize resources efficiently. Such adaptations often come at the cost of lowered reproductive rates, as organisms wait longer to reproduce so that they can grow larger and stronger, and produce fewer, larger offspring. Such populations are termed K-selected because they are adapted to thrive when the population is near its carrying capacity (K). Table 54.3 lists some of the typical features of K-selected populations. Examples of K-selected species include coconut palms, whooping cranes, whales, and humans. By contrast, in populations far below the carrying capacity, resources may be abundant. Costs of reproduction are low, and selection favors those individuals that can produce the maximum number of offspring. Selection here favors individuals with the highest reproductive rates; such populations are termed r-selected. Examples of organisms displaying ­r-­selected life history adaptations include dandelions, aphids, mice, and cockroaches. Most natural populations show life history adaptations that exist along a continuum ranging from completely r-selected traits to completely K-selected traits. Although these tendencies hold true in general, few populations are purely r- or K-selected and show all of the traits listed in table 54.3. These attributes should be treated as generalities, with the recognition that many exceptions exist.

TA B L E 5 4 . 3

r-Selected and K-Selected Life History Adaptations r-Selected Populations

Adaptation Population cycles are not as rare as traditionally thought; one review of nearly 700 long-term studies (25 years or more) of trends within populations found that cycles were not uncommon; nearly 30% of the studies—including birds, mammals, fish, and crustaceans—provided evidence of some cyclic pattern in population size through time, although most of these cycles are nowhere near as dramatic in amplitude as the hare–lynx cycles. In some cases, such as that of the snowshoe hare and lynx, densitydependent factors may be involved, whereas in other cases, density-independent factors, such as cyclic climatic patterns, may be responsible.

Resource availability affects life history adaptations As you have seen, some species usually maintain stable population sizes near the carrying capacity, whereas in other species population

K-Selected Populations

Age at first reproduction

Early

Late

Life span

Short

Long

Maturation time

Short

Long

Mortality rate

Often high

Usually low

Number of offspring produced per repro­ductive episode

Many

Few

Number of reproductions per lifetime

Few

Many

Parental care

None

Often extensive

Size of offspring or eggs

Small

Large

chapter

54 Ecology of Individuals and Populations 1231

Learning Outcomes Review 54.6

Density-dependent factors such as resource availability come into play particularly when population size is larger; density-independent factors such as natural disasters operate regardless of population size. Population density may be cyclic due to complex interactions such as resource cycles and predator effects. Populations with densitydependent regulation often are near their carrying capacity; in species with populations well below carrying capacity, natural selection may favor high rates of reproduction when resources are abundant. ■■

Can a population experience both positive and negative density-dependent effects?

54.7 Human

Population Growth

the carrying capacity of the habitats in which they lived and thus to escape the confines of logistic growth and re-enter the exponential phase of the sigmoidal growth curve. Responding to the lack of environmental constraints, the human population has grown explosively over the last 300 years. Although the birth rate has decreased over time, the death rate has decreased to a much greater extent. The difference between birth and death rates meant that the population grew as much as 2% per year, although the rate has now declined to 1.1% per year. A 1.1% annual growth rate may not seem large, but it has produced a current human population that is approaching 8 billion people (figure 54.24). At this growth rate, nearly 80 million people would be added to the world population in the next year, and the human population would double in 63 years. Both the current human population level and the projected growth rate have potentially grave consequences for our future.

Learning Outcomes

Humans exhibit many K-selected life history traits, including small brood size, late reproduction, and a high degree of parental care. These life history traits evolved during the early history of hominins, when the limited resources available from the environment controlled population size. Throughout most of human history, our populations have been regulated by food availability, disease, and predators. Although unusual disturbances, including floods, plagues, and droughts, no doubt affected the pattern of human population growth, the overall size of the human population grew slowly during our early history. Two thousand years ago, perhaps 130 million people populated the Earth. It took a thousand years for that number to double, and it was 1650 before it had doubled again, to about 500 million. In other words, for most of its existence, the human population was characterized by very slow growth. In this respect, human populations resembled many other species with predominantly K-selected life history adaptations.

Human populations have grown exponentially Starting in the early 1700s, changes in technology gave humans more control over their food supply, enabled them to develop superior weapons to ward off predators, and led to the development of cures for many diseases. At the same time, improvements in shelter and storage capabilities made humans less vulnerable to climatic uncertainties. These changes allowed humans to expand 1232 part VIII Ecology and Behavior

6

5 Billions of People

1. Explain how the rate of human population growth has changed through time. 2. Describe the effects of age distribution on future growth. 3. Evaluate the relative importance of rapid population growth and resource consumption as threats to the biosphere and human welfare.

7

4

3

Significant advances in public health

2

Industrial Revolution

1

Bubonic plague “Black Death”

4000 B.C.

3000 B.C.

2000 B.C.

1000 B.C.

0

1000

2000

Year

Figure 54.24 History of human population size. 

Temporary increases in death rate, even a severe one such as that occurring during the Black Death of the 1300s, have little lasting effect. Explosive growth began with the Industrial Revolution in the 1800s, which produced a significant, long-term lowering of the death rate. The current world population is approaching 8 billion, and if it continued to grow at the present rate, would double in 63 years.

?

Inquiry question Based on what we have learned

about population growth, what do you predict will happen to human population size?

occurring uniformly over the planet. Rather, most of the population growth is occur2020 ring in Africa and Asia (figure 54.25). By 2050 contrast, populations are actually decreas2100 5000 ing in some countries in Europe. The rate at which a population can be expected to grow in the future can be 4000 assessed graphically by means of a population pyramid, a bar graph displaying 3000 the numbers of people in each age category (figure 54.26). Males are conventionally shown to the left of the vertical age axis, 2000 females to the right. A human population pyramid thus displays the age composition of a population by sex. In most human 1000 population pyramids, the number of older females is disproportionately large compared with the number of older males, be0 Africa Asia Europe Latin America Northern Oceania cause females in most regions have a and America (including longer life expectancy than males. the Caribbean Australia) Viewing such a pyramid, we can predict demographic trends in births and Figure 54.25 Projected population growth for the rest of this century.  deaths. In general, a rectangular pyramid is Developed countries are predicted to grow little; almost all of the population increase will occur characteristic of countries whose popu­ in less-developed countries. lations are stable, neither growing nor shrinking. A triangular pyramid is characteristic of a country that Population pyramids show will exhibit rapid future growth because most of its population has not yet entered the childbearing years. Inverted triangles are charbirth and death trends acteristic of populations that are shrinking, usually as a result of Although the human population as a whole continues to grow rapsharply declining birth rates. idly at the beginning of the 21st century, this growth is not Population (in millions)

6000

Sweden

Figure 54.26 

Kenya

Comparison of Swedish and Kenyan population pyramids. Population

male female

100+ 95–99 90–94

pyramids from 2020 are graphed according to a population’s age distribution. Kenya’s pyramid has a broad base because of the great number of individuals below childbearing age. When the young people begin to bear children, the population will experience rapid growth. The Swedish pyramid exhibits many postreproductive individuals resulting from Sweden’s long average life span.

85–89 80–84 75–79 70–74 65–69 Age

60–64 55–59 50–54 45–49 40–44 35–39 30–34

?

25–29 20–24 15–19 10–14

Inquiry question  What will the population distributions look like in 20 years?

5–9 0–4 8

6

4

2

0

2

4

6

8

8

6

4

2

0

2

4

6

8

Percent of Total Population

chapter

54 Ecology of Individuals and Populations 1233

Humanity’s future growth is uncertain Earth’s rapidly growing human population constitutes perhaps the greatest challenge to the future of the biosphere, the world’s interacting community of living things. Humanity is adding 80 million people a year to its population—over a million every 5  days, 150 every minute! In more rapidly growing countries, the resulting population increase is staggering (table 54.4). India, for example, had a population of 1.05 billion in 2002; by 2050, its population likely will exceed 1.6 billion. A key element in the world’s population growth is its uneven distribution among countries. Of the billion people added to the world’s population in the 1990s, 90% live in developing countries (figure 54.27). The fraction of the world’s population that lives in industrialized countries is therefore diminishing. In 1950, fully one-third of the world’s population lived in industrialized countries; by 1996, that proportion had fallen to one-quarter; and by 2020, the proportion had fallen to one-sixth. In the future, the world’s population growth will be centered in the parts of the world least equipped to deal with the pressures of rapid growth. Rapid population growth in developing countries has had the harsh consequence of increasing the gap between rich and

TA B L E 5 4 . 4

11 10 World Population in Billions

Examples of population pyramids for Sweden and K ­ enya in 2016 are shown in figure 54.26. The two countries exhibit very different age distributions. The nearly rectangular population pyramid for Sweden indicates that its population is not expanding because birth rates have decreased and average life span has increased. The very triangular pyramid of Kenya, by contrast, results from relatively high birth rates and shorter average life spans, which can lead to explosive future growth. The difference is most apparent when we consider that only 18% of Sweden’s population is less than 15 years old, compared with more than 40% of all Kenyans. Moreover, the fertility rate (offspring per woman) in Sweden is 1.9; in Kenya, it is 3.5. As a result, Kenya’s population could double in 30 years, whereas Sweden’s will remain stable.

hf hi g

9 8

m diu me

World total

7 6 5

Developing countries

4 3 2 1 0 1900

Developed countries 1950

Time

2000

2050

Figure 54.27 Distribution of population growth. Most of the worldwide increase in population since 1950 has occurred in developing countries. The age structures of developing countries indicate that this trend will increase in the near future. World population in 2050 likely will be between 7.3 and 10.7 billion, according to a recent study. Depending on fertility rates, the population at that time will be increasing either rapidly or slightly, or in the best case, declining slightly. poor. Today, people that live in the industrialized world have a per capita income of $33,829, but those that live in developing countries have a per capita income of only $5,405. Furthermore, of the people in the developing world, about one-eighth of the population get by on less than $2 per day. No one knows whether the world can sustain today’s population of nearly 8 billion people, much less the far greater numbers expected in the future. As chapter 56 outlines, the world ecosystem is already under considerable stress. Despite our

Brazil (moderately developed)

Ethiopia (poorly developed)

Fertility rate

1.8

1.7

4.1

Population growth rate

0.72

0.67

2.56

Doubling time at current rate (years)

97

104

27

Infant mortality rate (per 1000 births)

5

16

36

Life expectancy at birth (years)

80

75

68

Per capita GDP (U.S. $)*

$59,800

$15,600

$2,200

Population < 15 years old (%)

18

21

40

1234 part VIII Ecology and Behavior

lity ferti

low fertility

A Comparison of 2019 Population Data in Developed and Developing Countries United States (highly developed)

*GDP, gross domestic product.

ty tili er

The population growth rate has declined The world population growth rate is declining, from a high of 2.0% in the period 1965–1970 to 1.1% in 2015. Nonetheless, because of the larger population, this amounts to an increase of 80 million people per year to the world population, compared with 53 million per year in the 1960s. Most countries are devoting considerable attention to slowing the growth rate of their populations, and there are genuine signs of progress. For example, since 1984, family planning programs in Kenya have succeeded in reducing the fertility rate from 8.0 to 3.5 children per couple, thus lowering the population growth rate from 4.0% per year to 2.3% per year. Because of these efforts, the global population may stabilize at about 8.9 billion people by the middle of the current century. How many people the planet can support sustainably depends on the quality of life that we want to achieve; there are already more people than can be sustainably supported with current technologies.

Consumption in the developed world further depletes resources Population size is not the only factor that determines resource use; per capita consumption is also important. In this respect, we in the industrialized world need to pay more attention to lessening the impact each of us makes because, even though the vast majority of the world’s population is in developing countries, the overwhelming percentage of consumption of resources occurs in the industrialized countries. Indeed, the wealthiest 20% of the world’s population accounts for 86% of the world’s consumption of resources and produces 53% of the world’s carbon dioxide emissions, whereas the poorest 20% of the world is responsible for only 1.3% of consumption and 3% of carbon dioxide emissions. Looked at another way, in terms of resource use, a child born today in the industrialized world will consume many more resources over the course of his or her life than a child born in the developing world. One way of quantifying this disparity is by calculating what has been termed the ecological footprint, which is the amount of productive land required to support an individual at the standard of living of a particular population through the course of his or her life. This figure estimates the acreage used for the production of food (both plant and animal), forest products, and housing, as well as the area of forest required to absorb carbon dioxide produced by the combustion of fossil fuels. As figure 54.28 illustrates, the ecological footprint of an individual in the United States is nearly eight times greater than that of someone in Nigeria. Based on these measurements, researchers have cal­culated­ that resource use by humans is now one-third greater than the amount that nature can sustainably replace. Moreover, consumption is increasing rapidly in parts of the developing world; if all

30 28 Acres of Land Required to Support an Individual at Standard of Living of Population

using an estimated 45% of the total biological productivity of Earth’s landmasses and more than one-half of all renewable sources of freshwater, between one-fourth and one-eighth of all people in the world are malnourished. We cannot reasonably expect to expand Earth’s carrying capacity indefinitely, and indeed we already seem to be stretching the limits.

26 24 22 20

19.9

18 16 14 11.6

12 10 8

6.9

6

4.1

4

2.9

2.5

India

Nigeria

2 0

USA

Germany

Brazil

Indonesia

Figure 54.28 Ecological footprints of individuals in different countries. An ecological footprint calculates how much

land is required to support a person through his or her life, including the acreage used for production of food, forest products, and housing, in addition to the forest required to absorb the carbon dioxide produced by the combustion of fossil fuels.

?

Inquiry question If the population of India is four times larger than the population of the United States, which country has a larger overall ecological footprint?

humans lived at the standard of living in the industrialized world, two additional planet Earths would be needed. Building a sustainable world is the most important task facing humanity’s future. The quality of life available to our children will depend to a large extent on our success in limiting both population growth and the amount of per capita resource consumption.

Learning Outcomes Review 54.7

For most of its history, the K-selected human population increased gradually. In the last 400 years, with resource control, the human population has grown exponentially; at the current rate, it would double in 63 years. A population pyramid shows the number of individuals in different age categories. Pyramids with a wide base are undergoing faster growth than those that are uniform from top to bottom. Growth rates overall are declining, but consumption per capita in the developed world is still a significant drain on resources. ■■

Which is more important, reducing global population growth or reducing resource consumption levels in developed countries?

chapter

54 Ecology of Individuals and Populations 1235

54.8 Pandemics

Health

and Human

Learning Outcomes 1. Describe how pandemics occur. 2. Explain how reproduction number determines whether an outbreak occurs or dies out. 3. Understand how disease-causing agents evolve adaptively.

Disease outbreaks can play a major role in determining population dynamics, in some cases possibly even causing the extinction of a species. For example, the chestnut blight, a pathogenic (diseasecausing) fungus, was accidentally introduced from Asia to North America in 1904. The American chestnut, formerly a dominant tree in eastern forests, has never recovered. Similarly, the rinderpest virus, introduced with a shipment of cattle from India, wiped out almost all of the cattle and many wildlife species—including Cape buffalo, giraffe, and wildebeest—in eastern and southern Africa in the late nineteenth century. In some cases, disease has even intentionally been introduced to reduce the population size of out-of-control non-native species. Rabbits, for example, were brought to Australia from Europe in the eighteenth century and in the absence of predators became a huge scourge, eating all available vegetation and damaging both native animal and sheep populations. When the myxoma virus was introduced by wildlife managers, more than 99% of the rabbits perished, although the population eventually rebounded. History shows that human populations, too, have been affected by disease outbreaks. The term pandemic is now commonly used to refer to widespread disease outbreaks extending over multiple countries and even continents, usually in reference to human populations (epidemic is the term for more localized outbreaks). Perhaps the most notorious human pandemic is the bubonic plague, caused by a bacterium, Yersinia pestis, and spread by fleas and through contact with infected individuals. This was one of a series of plagues caused by Y. pestis over the last several thousand years, with the “Black Death” being the worst. This pandemic killed a third of the population of Europe in the fourteenth century (see figure 54.24). In more recent times, the Spanish flu killed more than 50 million people in 1918–19, and the global toll of COVID-19 that erupted early in 2020 is still being calculated, with morethan four million lives lost as of the end of July 2021.

Pandemics result from pathogens infecting a host population lacking defenses A pandemic occurs when a pathogen becomes established in a population in which the host individuals do not have adequate immune system defenses (see chapter 50). This can occur when a population has no prior exposure to the pathogen, as occurs when

1236 part VIII Ecology and Behavior

a disease-causing agent moves from one species to another. We call these zoonotic diseases, and these emerging diseases are an increasing human health problem (see chapter 57). In other cases, such as influenza (chapter 26), the pathogen can evolve in such a way that the host individual’s immune system no longer defends against it.

Degree of disease spread determines whether a pandemic occurs An important factor determining the extent of a disease outbreak is the effectiveness of the pathogen in spreading from one host individual to another. If, on average, each infected individual infects one more individual, then the prevalence of the disease will remain constant in the population (the technical measure is termed the reproduction number, R0, pronounced “R naught”; figure 54.29). If, on the other hand, each individual passes on the pathogen to more than one other individual (R0 > 1), then the disease will increase; the greater the number of new individuals becoming infected per host, the more rapidly the disease will increase. For example, the R0 of SARS-CoV-2, the virus that causes COVID-19, was estimated to be somewhere between 2 and 3 at the outset of the pandemic. Conversely, if each individual produces less

R0 = 0.5

R0 = 1

R0 = 2

Figure 54.29 Reproduction number and pandemic outcome. The number of new individuals infected on average from

each infected individual (R0) determines whether an outbreak will occur or die out. A value of R0 > 1 will lead to increasing prevalence of the disease, whereas values